International Journal of Biological Macromolecules 31 (2002) 63
/69

www.elsevier.com/locate/ijbiomac

Synthesis of PHB by recombinant E. coli harboring an approximately

5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL
2209
T.V.N. Ramachander a, D. Rohini a, A. Belhekar b, S.K. Rawal a,*
a
Plant Tissue Culture, National Chemical Laboratory, Pune 411008, India
b
Catalysis Division, National Chemical Laboratory, Pune 411008, India

Received 10 May 2002; received in revised form 5 August 2002; accepted 9 August 2002

Abstract

An
/5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and
introduced into Escherichia coli . The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions.
The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85 /105. Purified PHB
granules were spherical with an average size of 1.1 mm and of stable configuration. DSC thermogram suggested high crystalline
nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 8C. Recombinant E. coli
cells preferentially utilized glycerol as the carbon source and accumulated 25
/28 times more PHB than the native S. aureofaciens .
# 2002 Elsevier Science B.V. All rights reserved.

Keywords: Polyhydroxybutyrate; Streptomyces ; Biodegradable plastics

1. Introduction pullulans [13] synthesize the water soluble polyester

Plastics derived from fossil fuels have become an
integral part of contemporary life [1]. However, their
non-degradable nature and accumulation as solid waste
pose a threat to the global environment. To satisfy the
environmental imperatives, novel non-petroleum based
biodegradable plastics having properties comparable
with conventional plastics are under development. These
include polylactides, polyglycolic acids, polyhydroxyalk-
anoates (PHAs), polysaccharides and their blends [2
/7].
PHAs, discovered by Lemoigne [8], are the most
studied biopolymers. Approximately 300 different types
of Gram negative and Gram positive bacteria synthesize
and accumulate PHAs as granular inclusions in the
cytoplasm. The PHAs accumulated as carbon and
energy storage materials or as a sink for the redundant
reducing power under limiting nutrient conditions [9
/
12]. Eukaryotic micro-organisms such as Aureobasidium
* Corresponding author. Tel.: /91-20-5893382x2219; fax: /91-20-
5893438
E-mail address: rawal@dalton.ncl.res.in (S.K. Rawal).
0141-8130/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 4 1 - 8 1 3 0 ( 0 2 ) 0 0 0 6 8 - 5
polymalic acid which is not synthesized by prokaryotes.
PHAs exhibit high molecular weights, thermoplastic
and/or elastomeric features besides other interesting
physical and mechanical properties. Because of the
biodegradable and biocompatible nature of PHAs, these
have applications in packaging and food industry,
medicine, pharmacy and agriculture or as raw materials
for the synthesis of enantiomerically pure chemicals.
Analytical techniques such as GC, NMR and FTIR
are used to identify, characterize and quantitate PHAs
[14
/23]. It has been shown that copolymers of various
PHAs possess superior thermal and mechanical proper-
ties than the homopolymers [2]. Medium chain length
PHAs have been found unsuitable for applications due
to their low melting points and low glass transition
temperatures [24,25]. A better understanding of the
physical and thermal properties of biopolymers should
help us design these with properties comparable to the
conventional plastics.
Among actinomycetes, Streptomyces sp. [26,27], Rho-
dococcus sp. [28
/30] and Nocardia asteriodes [31] are
reported to accumulate PHAs. In this paper, we report
64 T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63
/69
for the first time, the characterization data of the
biopolymer PHB (poly-3-hydroxybutyrate) synthesized
by recombinant E. coli harboring an approximately 5.0
kb genomic DNA fragment from S. aureofaciens NRRL
2209. Also, we report 25
/28 times higher PHB accu-
mulation by the recombinant E. coli than the native
organism S. aureofaciens .
2. Materials and methods
2.1. Biopolymer synthesis
A
/5.0 kb Sau3A I genomic DNA fragment from S.
aureofaciens NRRL 2209 was isolated and ligated to the
BamH I restriction site in pGEM 3Z plasmid vector
(Promega, USA). The ligation product was used to
transform E. coli JM109.
The recombinant E. coli was grown in BMG medium
(each liter of the medium contained: yeast extract 5.0
gm, bacterial peptone 5.0 gm, disodium hydrogen
phosphate 1.0 gm, magnesium sulfate 200 mg, glucose
10.0 gm, pH 7.2). Glucose in BMG medium was
replaced either with 1% glycerol (BMGL), 1% sucrose
(BMS) or 0.5% sodium acetate (BMSA). The cells were
also grown in Luria Bertani (LB) broth [32]. Cultures
were incubated at 37 8C with shaking at 200 rpm for 40
h. S. aureofaciens NRRL 2209 cells were grown in
production medium [26] (each liter of the medium
contained: yeast extract 2.5 gm, potassium chloride 3.0
gm, ammonium sulfate 5.0 gm, calcium carbonate 4.0
gm, soyabean dialysate 100 ml, glucose 20 gm, pH 6.8).
The cells were grown at 28 8C for 16
/18 h in baffled
flasks with continuous shaking at 200 rpm.
2.2. Recovery of the biopolymer from the cell
Modified method of Hahn et al. [33] was used to
recover the polymer from the recombinant E. coli .
Briefly, the cells were harvested by centrifugation at
2000 /g , washed twice with deionized water and freeze-
dried under vacuum. The lyophilized cell pellet was
shaken for 90 min at 37 8C with chloroform and 30%
sodium hypochlorite (1:1). The dispersion was centri-
fuged at 4000 /g , at room temperature for 10 min.
Lower chloroform phase that contained the solubilized
polymer was recovered and the polymer precipitated by
addition of 4 vol. of methanol.
2.3. Staining of the cells
Heat fixed bacterial smear was stained with 1.0%
aqueous solution of Nile blue A at 55 8C for 10 min.
The slide was washed with tap water to remove excess
stain and then with 8% aqueous acetic acid for 1 min.
The stained smear was again washed with water and
blotted dry. Before examination, the slide was remois-
tened with water and a cover slip was placed on the
smear [22]. The slides were observed under the oil
immersion lens of a Leica fluorescent microscope at an
excitation wavelength of 480 nm.
2.4. FTIR analysis
Biomass from a 100 ml cell culture was pelleted as
above, suspended in 10 ml distilled water and lyophi-
lized at /58 8C. A sample (2 mg) was thoroughly
mixed with KBr (sprectroscopic grade) and dried at
100 8C for 4 h [19]. FTIR spectrum was taken using a
Perkin Elmer (USA) model 1720 Fourier transform IR
spectrophotometer.
2.5. Gas chromatography analysis
Acidic propanolysis of dried cell pellet was carried out
according to Riis and Mai [17]. GC analysis was
performed using a Shimadzu GC 17-A gas chromato-
graph. A 0.32 mm diameter BP 1 capillary column
(J&W Scientific Co., USA) of 25 m length was used. The
analysis started at 80 8C for 5 min followed by 7 8C/
min rise in temperature to reach the final temperature of
200 8C. Nitrogen (5 ml/min) was used as the carrier gas.
Benzoic acid propyl ester was used as the internal
standard.
2.6. 1H NMR analysis of extracted polymer
Polymer recovered from the recombinant E. coli cells
was subjected to 1H NMR study. Standard PHB
solution (1% wt./vol.) was prepared in CDCl3. The
recovered polymer was also solubilized in CDCl3 at a
concentration of 5% (wt/vol.). 1H NMR spectra were
recorded using Bruker Ac200 at 24 8C [15].
2.7. Determination of molecular weight and molecular
weight distribution
A Waters model 150-CV gel permeation chromato-
graph at 40 8C was used for determining the molecular
weight and molecular weight distribution of the recov-
ered polymer (0.10% PHA in CHCl3). Monodisperse
polystyrene and chloroform were used as the molecular
weight standard and the mobile phase, respectively.
2.8. TG analysis of PHA
Thermogravimetric change of PHA sample was
analyzed using Rheometric TG analyser (Rheometric
Scientific, USA). The analysis was carried out under
nitrogen flow rate of 15 ml/min with a scanning rate of
10 8C/min.
 T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63
/69 65

2.9. Differential scanning calorimetry

To determine the morphological state of the polymer,

the melting temperature and the enthalpy of fusion were
measured using a Perkin Elmer differential scanning
calorimeter (DSC-7).

2.10. Scanning and transmission electron microscopy

Sample for scanning electron microscopy was pre-

pared by sonicating a purified suspension of PHB at 20
KHz. A drop of the suspension was dried on a brass
stub under an IR lamp and later coated with gold using
a Polaron sputter coater unit. SEM photographs were
taken using a LEICA Stereoscan 440 Scanning Electron
Microscope equipped with a Phoenix EDAX attach-
ment.
For TEM [16] the bacterial cell pellet was fixed for 2 h
in 0.1 M sodium cacodylate (pH 7.2) containing 2%
glutaraldehyde. Following fixation, the sample was
washed thrice with 0.1 M sodium cacodylate buffer
(pH 7.2), mixed with 1% OsO4 and 1% potassium
ferrocyanide in 0.1 M sodium cacodylate buffer (pH
7.2) for 2 h at room temperature. Specimen was washed
with water, poststained with 2% uranyl acetate for 1 h,
dehydrated in graded concentrations (70, 90 and 100% Fig. 1. Gas chromatograms of non-recombinant and recombinant E.
coli . Propyl ester of PHB and benzoic acid are represented by Peaks 1
v/v) of acetone and embedded in epon resin by incubat- and 2, respectively.
ing overnight at 70 8C. Thin section of 50 nm were cut,
stained with lead citrate and photographed with Tecnai
12 Electron microscope.

3. Results and discussion

3.1. Characterization of the PHA

Recombinant E. coli cells harboring
/5.0 kb geno-

mic DNA fragment from S. aureofaciens NRRL 2209
were initially screened for PHA accumulation by
fluorescence microscopy. The cells stained with Nile
blue A showed characteristic orange fluorescence when
observed under fluorescence microscope at an excitation
wavelength of 460 nm indicating PHA accumulation in
these cells (Figure not shown).
GC analysis of dried and esterified recombinant and
non-recombinant E. coli cells grown for 40 h revealed a
prominent peak corresponding to the propyl ester of 3-
hydroxybutyrate in the recombinant E. coli cells. This
peak was not observed in the non-recombinant E. coli
(Fig. 1).
FTIR analysis of the lyophilized recombinant E. coli
cell pellet showed two absorption bands at 1280 and
1730 cm 1 [19] corresponding to C /O and C /O
stretching groups, respectively. This confirmed the
presence of PHB in the recombinant cells (Fig. 2).
Fig. 2. FTIR spectra of recombinant E. coli cells containing PHB. The
absorption bands at 1280 and 1730 cm 1 correspond to C /O and C/
O of PHB, respectively.
The purity of the recovered polymer was further
confirmed by Lowry method [34] for the analysis of
protein content. Only trace amounts of residual protein
were detected. This purified PHB sample was next
subjected to 1D 1H NMR. Trimethylsilane was used as
the internal chemical shift standard. The spectrum (Fig.
3) revealed the presence of three groups of signals
characteristic of PHB homopolymer. The doublet at
1.3 ppm was attributed to the methyl group coupled to
66 T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63
/69

Fig. 3. 1H NMR spectra of the PHB recovered from the recombinant E. coli . The doublet, doublet of quadruplet and a multiplet at 1.3, 2.57 and 5.28
ppm are attributed to; the methyl group coupled to a proton, the methylene group adjacent to an asymmetric carbon atom bearing a single proton
and the methylene group, respectively. The signal at 7.25 ppm is characteristic of chloroform-d. Doublet of quadruplet of the methylene group is
indicative of the isotactic nature of PHB.

one proton; the doublet of quadruplet at 2.57 ppm to the

methylene group adjacent to an asymmetric carbon
atom bearing a single proton and the multiplet at 5.28
ppm to the methylene group. Chloroform-d gave a
chemical shift signal at 7.25 ppm. The signal multiplicity
by a proton as a quadruplet or octet in case of protons
of CH2 group is obtained due to proton coupling in
isotactic form unlike in syndiotactic where duplet signal
is obtained due to coupling. From these results, it was
concluded that recombinant E. coli cells exclusively
synthesized isotactic PHB homopolymer.
The thermodynamic properties of a polymer are
dependent on its number average molecular mass and
the bulk properties connected with large deformations Fig. 4. Gel permeation chromatography of the purified PHB sample.
are largely determined by weight average molecular Mw and Mn represent weight average and number average molecular
weights, respectively. The polydispersity index (Q ) [Mw/Mn] of 2.64
mass [16]. Molecular weight of PHB is dependent on the
indicative of the non-uniform PHB polymer chain formation within
physiological background or on the amount of PHA the cell cytoplasm.
synthase produced within the cell [30,35]. In the present
study, the purified PHB sample showed a weight average
Mol. wt. (Mw) to be 2.85 /105 and number average
Mol. wt. (Mn) to be 1.065 /105. The possible reason for
low molecular weight of PHB synthesized by the temperature and enthalpy of fusion of the polymer were
recombinant E. coli may be due to over expression of 173 8C and 85.0 J/g, respectively. The high enthalpy of
the PHA synthase gene [36,37] an inherent property of fusion suggests high crystalline nature of the recovered
the S. aureofaciens genes directing PHB synthesis. PHB which was calculated to be of 60
/65% assuming
The polydispersity index (Q ) (defined as Mw/Mn) of the enthalpy of fusion of 100% crystalline sample to be
2.64 (Fig. 4) was directly proportional to the hypo- 146 J/g [38]. The difference between the decomposition
chlorite treatment time. Where in Q increased with the and the melting temperatures of the recovered PHB was
increasing treatment time (data not shown). This also high enough to facilitate processing of the polymer.
indicated the non-uniform chain length of the polymer PHB granules coated with and without gold and
synthesized and accumulated within the recombinant observed under scanning electron microscope showed a
cells. uniform spherical shape and size with a stable config-
The recovered polymer rapidly degraded between 250 uration. The average diameter of the PHB granule was
and 340 8C with a peak at 295 8C (Fig. 5). The melting 1.1 mm (Fig. 6).
 T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63
/69
Fig. 5. Thermogravimetric analysis of the purified PHB sample. It
shows a rapid degradation of the recovered PHB between 250 and
340 8C with a peak at 295 8C.
Fig. 6. Scanning electron microscopy (SEM) of the gold coated PHB
granules. PHB granules of uniform spherical shape with a stable
configuration are seen. The size of the granule is
/1.1 mm. Bar
represents 1 mm.
3.2. Growth conditions
PHB accumulation by the recombinant E. coli
expressing the PHB biosynthesis genes from S. aureofa-
ciens was studied by growing these cells in a basal
medium supplemented with different carbon sources
(Table 1). The cells were also grown in LB medium.
Irrespective of the carbon source used the cells accumu-
late PHB through out the culture period reaching peak
value after 40 h of growth. Although acetate and glucose
are the precursors for acetyl CoA synthesis [39], these
supported a mere 4% accumulation of PHB on cell dry
weight basis. This was in total contrast with 70
/90%
PHB accumulation observed for recombinant E. coli
expressing the PHB biosynthetic genes from Ralstonia
67
Table 1
PHB accumulation by the recombinant E. coli in basal medium
containing glucose (BMG), glycerol (BMGL), sucrose (BMS) or
sodium acetate (BMSA), and in LB medium
Medium PHB (%)a
LB broth 1.090.5
BMG 4.091.2
BMGL 60.096.0
BMS 5.090.8
BMSA 4.091.1
a
Cell dry mass basis.
eutropha [40]. Sucrose likewise supported only 5% PHB
accumulation by the recombinant E. coli cells. Max-
imum PHB accumulation of 60
/66% was observed
when 1% glycerol was incorporated in the medium as
the sole carbon source. The possibility of the
/5 kb
genomic DNA fragment from S. aureofaciens carrying
genes which facilitate the conversion of glycerol to acetyl
CoA via either glycolysis or methylglyoxal pathway
(Fig. 7) and its subsequent incorporation into PHB
cannot be ruled out.
In contrast, the native S. aureofaciens NRRL 2209
accumulated a peak amount of 2.4% PHB after 16 h of
culture [26]. The PHB content in the cells declines
hereafter, to undetectable levels by the 30 h of culture
Fig. 7. PHB synthesis by Methylglyoxal pathway. 1.1.1.6, 2.7.1.29,
4.2.3.3 (earlier 4.2.99.11) and 1.2.1.3 are the IUBMB nomenclature for
the enzymes Glycerol dehydrogenase, Glycerone kinase, Methylglyox-
al synthase and Aldehyde dehydrogenase, respectively.
68 T.V.N. Ramachander et al. / International Journal of Biological Macromolecules 31 (2002) 63
/69
period. The metabolism of accumulated PHB may be
due to the action of unspecific esterases or lipases [41,42]
or depolymerases [2] present within the S. aureofaciens
cells. Thus, heterologous expression of the S. aureofa-
ciens PHB biosynthetic genes in E. coli resulted in about
25
/28 times higher PHB accumulation as also in
preferential utilization of glycerol as the carbon source.
TEM of the 40
/42 h cell cultures showed presence of
large PHB granules in the cell cytoplasm. The polymer
granules grow so large that gross deformation of the cell
shape occurs (Fig. 8), however, most of the cells were
found to be intact.
4. Conclusions
For the first time a approximately 5.0 kb DNA
fragment from S. aureofaciens NRRL 2209 responsible
for PHB synthesis was isolated. This fragment upon
cloning and expression in E. coli supported 25
/28 times
more PHB in recombinant E. coli than the native
organism when glycerol was supplied as the sole carbon
source. The recovered PHB was found to be an isotactic,
crystalline homopolymer with a granule size of 1.1 mm.
DSC and TGA results show a shift in the thermal
degradation temperature pattern of the polymer which is
found to be at 295 8C. Extraction by sodium hypo-
chlorite and subsequent GC analysis showed that the
polymer was of high purity. Molecular weight of the
PHB synthesized and accumulated by E. coli was found
to be around 3/105 by GPC.
Fig. 8. Transmission electron microscopy of the recombinant E. coli
cells showing PHB as cytoplasmic inclusion bodies. Bar represents 1
mm.
Acknowledgements
TVNR and RD thank Council of Scientific and
Industrial Research and University Grants Commission,
respectively, for the grant of their Research fellowships.
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