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Evaluation of promoters for gene expression in polyhydroxyalkanoate-

producing Cupriavidus necator H16

Article in Applied Microbiology and Biotechnology · March 2011

DOI: 10.1007/s00253-011-3100-2 · Source: PubMed

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 Appl Microbiol Biotechnol (2011) 89:1527–
DOI 10.1007/s00253-011-3100-2
APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Evaluation of promoters for gene expression
in polyhydroxyalkanoate-producing
Cupriavidus necator H16
Toshiaki Fukui • Kei Ohsawa • Jun Mifune •
Izumi Orita • Satoshi Nakamura
Received: 14 October 2010 / Revised: 28 November 2010 / Accepted: 29 November 2010 / Published online: 29 January 2011
Ⓒ Springer-Verlag 2011
Abstract Five kinds of promoters were evaluated as tools
for regulated gene expression in the PHA-producing
bacterium Cupriavidus necator. Several broad-host-range
expression vectors were constructed by which expression
of a reporter gene gfp was controlled by Plac, Ptac, or PBAD
derived from Escherichia coli, or promoter regions of
phaC1 (PphaC) or phaP1 (PphaP) derived from C. necator.
Then, the gfp-expression profiles were determined in C.
necator strains harboring the constructed vectors when the
cells were grown on fructose or soybean oil. Plac, Ptac,
PphaC, and PphaP mediated constitutive gene expression,
among which Ptac was the strongest promoter. lacI-Ptac was
not thoroughly functional even after addition of isopropyl-
β- D-thiogalactopyranoside (IPTG), probably due to
inability of
C. necator to uptake IPTG. Gene expression by araC-PBAD
could be regulated by varying L-arabinose concentration in
the medium, although P(3HB) production rate was slightly
decreased in the recombinant. phaR-PphaP exhibited an
expression profile tightly coupled with P(3HB) accumula-
tion, suggesting application of the vector harboring phaR-
PphaP for gene expression specific at the PHA- biosynthesis
phase. The properties of these promoters were expected to
be useful for effective engineering of PHA biosynthesis in
C. necator.
Keywords Polyhydroxyalkanoate . Poly
(3-hydroxybutyrate) . Promoter . Cupriavidus necator
T. Fukui (*) : K. Ohsawa : J. Mifune : I. Orita : S.
Nakamura
Department of Bioengineering, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of Technology,
B-37 4259 Nagatsuta, Midori-ku,
Yokohama 226-8501, Japan
e-mail: tfukui@bio.titech.ac.jp
Introduction
Recent global concern for environmental protection has
provided impetus for utilization of renewable natural
resources. Polyhydroxyalkanoates (PHAs) are a class of
bio-based polymers produced by a variety of bacteria from
biomass as an intracellular carbon- and energy-storage
material under unbalanced growth conditions. As PHAs are
biopolyesters exhibiting biodegradability after extraction
from the cells, they have drawn increasing attention as
possible alternatives for petroleum-based polymers
(Madison and Huisman 1999; Philip et al. 2007; Sudesh
et al. 2000).
The best-characterized PHA is poly[(R)-3-hydroxybutyrate]
[P(3HB)] produced by diverse bacteria, and a Gram-
negative bacterium Cupriavidus necator (formerly Ralstonia
eutropha) has been known to be an efficient producer of
P(3HB). The genes of the three enzymes for P(3HB)
biosynthesis, β- ketothiolase (PhaA), NADPH-dependent
acetoacetyl-CoA reductase (PhaB1), and PHA synthase
(PhaC1) are clustered in the chromosome 1 of C. necator
as phaC1-A-B1 (Pohlmann et al. 2006). The application
range of bacterial P(3HB) is restricted due to the low
resistance to stress caused by the brittle properties with low
elongation before breaking; however, copolymerization of
3HB with a second monomer unit can improve its thermal
and mechanical properties (Madison and Huisman 1999;
Sudesh et al. 2000). Previous studies have demonstrated
that C. necator can biosynthesize the 3HB-based
copolyesters containing (R)-3-
hydroxyvalerate, 4-hydroxybutyrate, 3-hydroxypropionate,
or (R)-4-hydroxyvarelate when the related alkanoates or
hydroxyalkanoates were fed as precursors for the desired
second units (Steinbüchel and Valentin 1995). Moreover,
in the last two decades, C. necator has been used as a host
for functional analyses of PHA-biosynthesis genes and
as a
 15 Appl Microbiol Biotechnol (2011) 89:1527–

platform for molecular breeding aiming efficient profiles of gene expression by these promoters have not
production of PHAs. For example, phaC genes derived been investigated.
from various sources were introduced into C. necator PHB-
4, a PHA- negative mutant (Schlegel et al. 1970), to
evaluate PHA- polymerization activity of the translated
synthases (Fukui and Doi 1997; Hustede et al. 1992;
Liebergesell et al. 2000; Liebergesell and Steinbüchel
1992, 1993; Matsusaki et al. 1998; Pieper and Steinbüchel
1992; Sudesh et al. 1998; Timm and Steinbüchel 1992).
Modification of the metabolic flux of gluconate to P(3HB)
in C. necator wild-strain H16 was conducted by expressing
genes encoding enzymes in the pentose-phosphate pathway
(Lee et al. 2003). It has been demonstrated that
recombinant strains of PHB-4 harboring phaC from
Aeromonas caviae or the mutant gene were excellent
producers of poly((R)-3-hydroxybutyrate-co-(R)-3-
hydroxyhexanoate) [P(3HB-co-3HHx)] from vegetable oils
(Fukui and Doi 1998; Kahar et al. 2004; Tsuge et al. 2007).
These recombinant strains also possessed ability to
synthesize copolyesters of 3HB and (R)-3-hydroxy-4-
methylvalerate when 4-methylvalerate was added into the
medium as a precursor (Tanadchangsaeng et al. 2009). Our
research group has also reported construction of artificial
metabolic pathways in C. necator for biosynthesis of
P(3HB-co-3HHx) and poly ((R)-3-hydroxybutyrate-co-3-
hydroxypropionate) from unre- lated carbon sources (Fukui
et al. 2002; Fukui et al. 2009) and engineering of pha
operon on the chromosome 1 of C. necator H16 for
efficient biosynthesis of P(3HB-co-3HHx)
with favorable 3HHx composition (7∼10 mol%) from
soybean oil (Mifune et al. 2008; Mifune et al. 2010).
In order to achieve effective metabolic engineering of
PHA-producing bacteria, the genes for PHA biosynthesis
or the related carbon and energy metabolisms should be
appropriately expressed or silenced without negative
impacts on other cellular functions such as growth ability.
Unlike strains such as Escherichia coli, Bacillus subtilis, or
other well-studied microorganisms, information regarding
promoters for regulated gene expression in C. necator has
been quite limited. The native region upstream of phaC in
interest was usually employed as a promoter for the
heterologous expression in C. necator. Delamarre et al.
have constructed recombinant strains of C. necator PHB-4
expressing phaC1Ps from Pseudomonas sp. 61-3 along with
phaAB1Cn under the control of promoter regions of acetoin
dehydrogenase system derived from C. necator (Delamarre
and Batt 2006). The recombinant strains accumulated
PHAs with altered compositions depending on acetoin
concentra- tion added into the medium, although the
overall yield of PHAs was lower than 10 wt.%. It was
reported that expression of heterologous genes located
downstream of E. coli BAD promoter was inducible in C.
necator by addition of L-arabinose (Fukui et al. 2002;
Fukui et al. 2009). However, in these studies, detailed
 Appl Microbiol Biotechnol (2011) 89:1527–
This study focused on evaluation of five kinds of respectively. phaR genes along with the
promoters as tools for gene expression aiming effective
metabolic modification for PHA biosynthesis in C.
necator.

Materials and methods

Bacterial strains and cultivation conditions

C. necator strains H16 (wild, DSM428) and PHB-4 (PHA-

deficient mutant (Schlegel et al. 1970), DSM541) and
their derivatives were cultivated in a nutrient-rich medium
as described previously (Fukui and Doi 1997; Fukui et al.
1998). E. coli DH5α and S17-1 (Simon et al. 1983) were
grown at 37°C in an Luria–Bertani (LB) medium. Kana-
mycin (50 mg/l for E. coli and 100 mg/l for C. necator
strains) or ampicillin (50 mg/l) was added to the medium
when necessary.

Construction of expression vectors

General genetic manipulations were performed according

to the standard procedures. Sequences of all the
oligonucleo- tides used for PCR in this study will be
available upon request. A 855-bp fragment including a
gene of green fluorescent protein (gfp) and the flanking
restriction sites was amplified from pUTmini-Tn5gfp
(de Lorenzo et al. 1990) with a primer set gfp-N/gfp-C,
followed by insertion into the HincII site of pUC118.
gfp was excised from the resulting plasmid, pUCgfp2, by
EcoRI, EcoRI–SalI, or EcoRI–HindIII digestion, and then
used as a reporter gene. pBBad-gfp (BAD promoter) was
constructed by inserting gfp excised from pUCgfp2 by
EcoRI–SalI digestion into pBBad (Fukui et al. 2009) at
the corresponding sites. pBLac- gfp (lac promoter) was
constructed by ligation of the gfp-rrnB1T1T2 terminator
(TrrnB) fusion excised from pBBad-gfp by EcoRI–SspI
digestion with a plasmid back- bone of pBBR1-MCS2
(Kovach et al. 1995) prepared by
EcoRI−SmaI digestion.
pBPC-gfp (phaC1 promoter) and pBPP-gfp (PhaP1
promoter) were constructed as follows. The upstream
regions of phaC1 (466 bp) and phaP1 (252 bp) were
amplified from C. necator genomic DNA using primer
sets PphaC-5/PphaC-3 and PphaP-5/PphaP-3,
respectively, and the amplified fragments were subcloned
into pBAD24 (Guzman et al. 1995) at the BamHI site.
The promoter- multiple cloning sites (MCS)-TrrnB fusions
were excised from the resulting plasmids by SspI
digestion, and then respectively ligated with a plasmid
backbone of pBBR1- MCS2 prepared by SspI digestion,
to obtain empty vectors pBPC and pBPP. Further
insertion of gfp at EcoRI–SalI sites of pBPC and EcoRI
site of pBPP gave pBPC-gfp and pBPP-gfp,
 Appl Microbiol Biotechnol (2011) 89:1527– 15
upstream regions of different lengths (258 and 232 bp) result was represented as a fluores- cence unit per RCM in
were amplified from C. necator genomic DNA using 100 ml culture broth (FU/g-cells).
primer sets PlphaR-5/phaR-3 and PsphaR-5/phaR-3,
respectively. The amplified long and short fragments were
inserted into pBPP-gfp at the SspI site, resulting in the
construction of pBphaRlPP-gfp and pBphaRsPP-gfp
(phaR, phaP1 promoter), respectively.
For construction of pBTac-gfp (tac promoter) and
pBlacITac-gfp (lacI, tac promoter), tac promoter-MCS-
TrrnB and lacI-tac promoter-MCS-TrrnB regions were
amplified from pJAK14 (ATCC77289) with primer sets
Ptac-5/rrnB and lacIq/rrnB, respectively. The amplified
fragments were then ligated with the plasmid backbone of
pBBR1-MCS2 prepared by SspI digestion. gfp was
inserted into the empty vectors pBTac and pBlacITac at the
EcoRI site and EcoRI/HindIII sites, to obtain pBTac-gfp
and pBlacITac-gfp, respectively.
Transconjugation of C. necator with E. coli S17-1 (Simon
et al. 1983) harboring the constructed gfp-expression
vectors was performed as described previously (Fukui and
Doi 1997).

PHA production and gfp expression

PHA production and gfp expression by C. necator strains

were carried out on a reciprocal shaker (115 strokes/min)
at 30°C for 72 h in 500 mL flasks with 100 mL of a
nitrogen- limited mineral salts medium (Fukui and Doi
1997; Kato et al. 1996). A filter-sterilized solution of
fructose was added to the medium at a final concentration
of 0.5% (w/v). Soybean oil was supplied directly at 1%
(v/v). For maintenance of broad-host-range plasmids,
kanamycin was added to the culture medium at a
concentration of
100 mg/L. When needed, L-arabinose (0.01% or 0.1%
(w/v)) or isopropyl-β-D-thiogalactopyranoside (IPTG)
(2 mM) was added into the culture broth at 12 h to induce
gene expression.
The culture broth at each cultivation time was divided to
90 and 10 ml. The cells harvested from the 90 ml broth by
centrifugation (8,000×g for 10 min at 4°C) were washed
with cold deionized water, and then freeze-dried to
measure cellular PHA content and residual cell mass
(RCM). The cellular PHA contents were determined by gas
chromatogra- phy after methanolysis of the polyesters
within dried cells as described previously (Kato et al. 1996).
The expression of gfp within the cells was evaluated by
measuring the cellular fluorescence. The cells in the
remaining 10 ml broth were harvested by centrifugation
(8,000×g for 10 min at 4°C) and resuspended in the same
volume of 50 mM Tris–HCl (pH 7.5). After incubation
on ice for 10 min, the fluorescent emission was measured
at 520 nm with excitation at 488 nm using a fluorescence
spectrophotometer PF-5300PC (Shimadzu, Japan). The
 15 Appl Microbiol Biotechnol (2011) 89:1527–
Results PphaC (H16/pBPC-gfp) were almost constant

Construction of gfp-expression vectors

Five kinds of promoters, lac (Plac), tac (Ptac), and BAD

promoters (PBAD) derived from E. coli, and promoter
regions of phaC1 (PphaC) and phaP1 (PphaP) derived from
C. necator were evaluated as promoters for gene
expression in C. necator. The expression vectors
containing gfp located between one of the promoters and
a rrnB1T1T2 terminator region (TrrnB) were constructed
based on a broad-host-range plasmid pBBR1-MCS2
(Table 1). The general structure of the gfp-expression
vectors was illus- trated in Fig. 1. The reporter gene gfp
was derived from pUTmini-Tn5gfp, and all the gfp-
expressing vectors exam- ined here harbored a common
ribosome-binding sequence located 8-bp upstream from
the start codon of gfp. pBlacITac-gfp, pBBad-gfp, and
pBphaRlPP-gfp and pBphaRsPP-gfp contained the
specific regulator gene lacI, araC, and phaR, respectively,
with direction opposite to that of the promoters. The
regions upstream of phaR in
pBphaRlPP-gfp and pBphaRsPP-gfp include a −24/−12
promoter region and upstream activation sequence
(UAS)
for phaR expression (Pötter et al. 2002). The length of the
upstream region in pBphaRlPP-gfp was 26-bp longer than
that in pBphaRsPP, as shown in Fig. 2.
The constructed plasmids were then used to transform
C. necator H16 and PHB-4. The resulting recombinant
strains were cultivated in a nitrogen-limited mineral salt
medium containing fructose or soybean oil as a sole
carbon source at 30°C, and the expression of gfp mediated
by the respective promoter was determined along with the
cell growth and P(3HB) production. The results with the
cells grown on 0.5% fructose for 36 h and 1.0% soybean
oil for 48 h are summarized in Table 2.

Expression profiles by Plac, Ptac, and PphaC in C. necator

on fructose

Figure 3a shows the time-dependent changes of the cell

growth, P(3HB) production, and gfp expression by C.
necator H16 harboring pBLac-gfp, pBTac-gfp,
pBlacITac- gfp, or pBPC-gfp cultivated on fructose for 72
h. As the cell growth and P(3HB) accumulation were very
similar among these strains, the black and gray bars in
Fig. 3a indicate the averages of residual cell masses
(RCMs) and P(3HB) production, respectively. RCMs
reached to maximum (approx. 0.8 g/l) after 24 h
cultivation, and the cells started to accumulate P(3HB) at
the late-growth phase. P(3HB) production reached to
maximum, approx. 1.3 g/l corresponding to 62 wt.% of dry
cell mass, at 36 h and kept the maximum level until 72 h.
The gfp-expression levels by Plac (H16/ pBLac-gfp) and
 Appl Microbiol Biotechnol (2011) 89:1527– 15
Table 1 Bacterial strains and plasmids used in this study

Strains or plasmids Relevant characteristics Source or reference

Strains
Cupriavidus necator
H16 Wild type DSM428
PHB-4 phaC− DSM541
Escherichia coli
DH5α deoR endA1 gyrA96 hsdR17 (rK− mK+) recA1 relA1 supE44 thi-l Δ(lacZYA-argFV169) Clontech
f80ΔlacZdM15 F- 1-
S17-1 thi pro hsdR recA; chromosomal RP4; Tra+; Tmpr Str/Spcr Simon et al. 1983
Plasmids
pUC118 Plac, lacZα, Ampr Takara
pBAD24 PBAD, araC, TrrnB, Ampr Guzman et al. 1995
pUTmini-Tn5gfp Mini-Tn5, gfp, mob, Ampr, Tetr de Lorenzo et al. 1990
pBBR1-MCS2 Broad-host-range vector, mob, Plac, lacZα, Kanr Kovach et al. 1995
pJAK14 Broad-host-range vector, mob, Ptac, lacI, Kanr ATCC77289
pBBad pBBR1-MCS2 derivative; PBAD, TrrnB, araC Fukui et al. 2009
pBLac-gfp pBBR1-MCS2 derivative; gfp, TrrnB This study
pBTac-gfp pBBR1-MCS2 derivative; Ptac, gfp, TrrnB This study
pBlacITac-gfp pBBR1-MCS2 derivative; lacI, Ptac, gfp, TrrnB This study
pBBad-gfp pBBad derivative; gfp This study
pBPC-gfp pBBR1-MCS2 derivative; PphaC, gfp, TrrnB This study
pBPP-gfp pBBR1-MCS2 derivative; PphaP, gfp, TrrnB This study
pBphaRlPP-gfp pBBR1-MCS2 derivative; phaR along with 258-bp upstream region, PphaP, gfp, TrrnB This study
pBphaRsPP-gfp pBBR1-MCS2 derivative; phaR along with 232-bp upstream region, PphaP, gfp, TrrnB This study

around 800 and 1,000 FU/g-cells, respectively, throughout the
cultivation, demonstrating that Plac and PphaC mediated
constitutive gene expression in C. necator. Ptac (H16/
pBTac-gfp) showed 1.5–2-fold higher expression of gfp when
compared to Plac and PphaC. Although the fluorescence was
highest at the late-growth phase and gradually decreased
after the PHA-biosynthesis phase, Ptac acted as the
strongest promoter among the promoters examined in this
study with
nearly constitutive expression profile. It is well known that
gene expression under the control of Plac or Ptac in lacIq
strains of E. coli is inducible by IPTG that binds and
inhibits LacI repressor. We therefore examined the effect of
IPTG on the gfp expression by Plac in C. necator, but the
expression profile was not changed at all when compared to
that without addition of IPTG. The lack of inducibility by
IPTG was likely due to the absence of LacI homolog in C.
necator genome (Pohlmann et al. 2006). pBlacITac, in
which lacI was located upstream of Ptac, was further
constructed aiming to develop an IPTG-inducible
expression system in this bacterium. However, no GFP
fluorescence was observed within the cells of
H16/pBLacITac-gfp even after addition of IPTG, indicat-
ing that IPTG was incapable of acting as an inducer for
gene expression by Ptac in C. necator.

Fig. 1 General structure of the broad-host-range gfp-expression

vectors used in this study. Kanr, mob, rep are derived from pBBR1-
MCS2. MCS (multiple cloning site) is derived from pBBR1-MCS2
(pBLac-gfp), pBAD24 (pBBad-gfp, pBPC-gfp, pBPP-gfp,
pBphaRlPP-gfp, and pBphaRsPP-gfp), or pJAK14 (pBTac-gfp and
pBlacITac-gfp). TrrnB (rrnB1T1T2 terminator) is derived from
pBAD24 ( pBLac-gfp, pBBad-gfp, pBPC-gfp, pBPP-gfp,
pBphaRlPP-gfp, and pBphaRsPP-gfp), or pJAK14 (pBTac-gfp and
pBlacITac-gfp). gfp along with RBS (ribosome-binding sequence) is
derived pUTmini-Tn5gfp
Expression profiles by PBAD in C. necator on fructose
Our previous studies have demonstrated that broad-host-
range plasmids harboring P BAD along with araC regulator
gene were also functional in C. necator as L-arabinose-
inducible expression vectors (Fukui et al. 2002; Fukui et al.
2009). In this study, we further determined the detailed
profile of gene expression under the control of araC-PBAD
 15 Appl Microbiol Biotechnol (2011) 89:1527–
in this bacterium grown on fructose. Figure 3b shows
 Appl Microbiol Biotechnol (2011) 89:1527– 15
Fig. 2 Nucleotide sequences of
the junction region between gfp
and phaR in pBphaRlPP-gfp
and pBphaRsPP-gfp

averages of RCMs (black bar) and P(3HB) production
(gray bar) of H16/pBBad-gfp with addition of 0%, 0.01%,
and 0.1% of L-arabinose at 12 h. The maximum RCMs and
P (3HB) production were similar to those of the cells
harboring the other pBBR1-based expression vectors
examined above,
whereas the rates of cell growth and P(3HB) accumulation
were slightly reduced; RCMs and P(3HB) production
reached to maximum at 36 and 48 h, respectively. The gfp
expression by PBAD was tightly regulated by L-arabinose
concentration. The fluorescence was negligible at every
point during the

Table 2 Cell growth, P(3HB) biosynthesis, and GFP fluorescence in recombinant strains of C. necator expressing gfp under the control of
various promotersa

Strain/Plasmid Inducer 0.5% Fructose, 36 h 1.0% Soybean oil, 48 h

RCMb (g/l) P(3HB)c (g/l) GFPd (FU/g-cells) RCMb (g/l) P(3HB)c (g/l) GFPd (FU/g-cells)

H16/pBBR1-MCS2 0.79 1.17 19 0.74 2.00 38

H16/pBLac-gfp 0.78 1.23 824 0.68 1.73 590
IPTG 2 mM 0.77 1.27 861
H16/pBTac-gfp 0.75 1.36 1,770 0.78 2.13 1940
H16/pBlacITac-gfp IPTG 2 mM 0.85 1.22 41
H16/pBBad-gfp Arabinose 0% 0.82 0.86 64 0.52 1.48 76
Arabinose 0.01% 0.71 0.86 210 0.65 1.64 174
Arabinose 0.1% 0.74 0.70 819 0.63 1.71 854
H16/pBPC-gfp 0.73 1.31 1020 0.71 1.84 699
H16/pBPP-gfp 0.72 1.42 965 0.76 2.10 1040
H16/pBphaRsPP-gfp 0.93 1.50 410 0.74 2.78 670
H16/pBphaRlPP-gfp 0.79 0.44 655
PHB-4/pBPP-gfp 0.69 0 1130
PHB-4/pBphaRsPP-gfp 0.60 0 23
PHB-4/pBphaRlPP-gfp 0.22 0 55

a
Cells were cultivated at 30˚C in a nitrogen-limited mineral salts medium containing 0.5% (w/v) fructose for 36 h or 1% (v/v) soybean oil for 48 h
b
Residual cell mass
c
Determined by methanolysis and GC analysis of the dried cells
d
Fluorescent units per gram residual cell mass in 100 ml culture broth
 15 Appl Microbiol Biotechnol (2011) 89:1527–

2000 2.0 2000 2.0

1500 1.5 1500 1.5

(g/L) P(3HB)
(g/L) P(3HB)
Residual cell mass

Residual cell mass

GFP (FU/g-

GFP (FU/g-
1000 1.0 1000 1.0

500 0.5 500 0.5

0 0.0 0 0.0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)

Fig. 3 Time-dependent changes of gfp expression by lac, tac, and

open inverted triangles, H16/pBLac-gfp with addition of 2 mM IPTG
phaC promoters in recombinant strains of C. necator H16 grown on
at 12 h; open squares, H16/pBTac-gfp (Ptac); and open diamonds,
0.5% fructose. Black bars, residual cell mass (grams per liter); gray
H16/ pBlacITac-gfp (lacI, Ptac) with addition of 2 mM IPTG at 12 h.
bars, P(3HB) production (grams per liter); and line plots, GFP
b H16/ pBBad-gfp (PBAD) with addition of L-arabinose at 12 h. Open
fluorescence (fluorescent unit/g-residual cell mass [FU/g-cells]). a
circles, no addition of L-arabinose; open triangles, 0.01% L-arabinose;
Open circles, H16/pBPC-gfp (PphaC); open triangles, H16/pBLac-gfp
and open squares, 0.1% L-arabinose
(Plac);

cultivation when L-arabinose was absent in the medium, vector. We

and the expression was gradually increased after addition
of the inducer at 12 h with a roughly constant rate
throughout the cultivation. The rate of fluorescence
increased and final level of the fluorescence at 72 h became
higher with increased concentrations of L-arabinose.

Expression profiles by PphaP in C. necator on fructose

It has been reported that PhaR autoregulator binds to PphaP

region to repress the transcription of phaP1 under cultiva-
tion conditions not permissible for PHA biosynthesis and
moves to PHA granules after formation of nascent
granules, resulting in derepression of the transcription of
phaP1 coupled with PHA biosynthesis in C. necator
(Pötter et al. 2002; Pötter and Steinbüchel 2005; York et al.
2002). We therefore consider applying PphaP as a useful
promoter that can mediate gene expression specific to the
PHA- biosynthesis phase. pBPP-gfp containing PphaP alone
without phaR was constructed and used to transform
C. necator H16 and PHB-4, then the relationship between
the gene expression by PphaP and P(3HB) accumulation on
fructose was examined. As shown in Fig. 4a, b, both
transformants exhibited similar growth profiles, and the
GFP fluorescence was within a level of 500–1,000 (FU/g-
cells) irrespective of P(3HB) accumulation. The sole
region of PphaP mediated constitutive gene expression with
moderate strength, like PphaC and Plac. The reason for the
independency of expression by PphaP on P(3HB) biosyn-
thesis was assumed to be due to insufficient amount of
PhaR regulator, expressed from the endogenous phaR
gene, to repress all PphaP regions on the multi-copy
 Appl Microbiol Biotechnol (2011) 89:1527– 15
therefore designed a new expression vector pBphaRlPP-
gfp, containing phaR and a 258 bp-region upstream of
phaR to provide larger amount of PhaR regulator. As
expected, the GFP fluorescence was detected only in the
wild-strain H16 and not at all in the PHA-negative mutant
PHB-4 (Fig. 4c, d). The expression of gfp was enhanced
with increasing P(3HB) accumulation and then slightly
decreased after the PHA-biosynthesis phase, displaying
tight coupling of the gene expression with P(3HB)
biosynthesis. The maximum GFP fluorescence was
similar to that in H16/pBPP-gfp not containing phaR.
However, the lag-phases of cell growth and P(3HB)
accumulation were significantly prolonged for
H16/pBphaRlPP-gfp (Fig. 4c, d, and Table 2). In order to
vary expression level of phaR, pBphaRsPP-gfp was
further constructed in which phaR upstream region was 26
bp shorter than that in pBphaRlPP- gfp. With the new
plasmid, the expression of gfp was again detected only in
H16 but not in PHB-4 (Fig. 4e, f, and Table 2). The
cell growth and P(3HB) accumulation in H16/pBphaRsPP
were comparable with the strain harboring pBPP-gfp,
whereas the maximum GFP fluorescence was lower
(approx. 500 FU/g-cells). The results demonstrated that
pBphaRsPP was useful as a unique expression vector
enabling autoregulation of gene expression tightly linked
with P(3HB) biosynthesis, although the level of gene
expression was not so high.

Expression profiles by Plac, Ptac, PphaC, PphaP, and PBAD

in C. necator on soybean oil

We further investigated the expression profiles of gfp by the

promoters in C. necator grown on 1.0% soybean oil
 15 Appl Microbiol Biotechnol (2011) 89:1527–

2000
2.0 2000 2.0

1500 1.5 1500 1.5

(g/L) P(3HB)
(g/L) P(3HB)

Residual cell mass

Residual cell mass
GFP (FU/g-

GFP (FU/g-
1000 1.0 1000 1.0

500 0.5 500 0.5

0 0.0 0 0.0
0 20 40 60 80 0 20 40 60 80
Time (h)
Time (h)

2000
2.0 2000 2.0

1500 1.5 1500 1.5

(g/L) P(3HB)
Residual cell mass
(g/L) P(3HB)
Residual cell mass
GFP (FU/g-

GFP (FU/g-

1000 1.0 1000 1.0

500 0.5 500 0.5

0 0.0 0 0.0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)

2000 2.0
2.0 2000

1500 1.5
1.5 1500
(g/L) P(3HB)

(g/L) P(3HB)
Residual cell mass

Residual cell mass

GFP (FU/g-

GFP (FU/g-

1000 1.0
1.0 1000

500 0.5
0.5 500

0 0.0
0.0 0
0 20 40 60 80 0 20 40 60 80
Time (h) Time (h)

Fig. 4 Time-dependent changes of gfp expression by phaP promoter

in recombinant strains of C. necator H16 and PHB-4 grown on 0.5% (PphaP), b PHB-4/pBPP-gfp, c H16/pBphaRlPP-gfp (phaR along with
fructose. Black bars, residual cell mass (grams per liter); gray bars, 258 bp-upstream region, PphaP), d PHB-4/pBphaRlPP-gfp, e H16/
P(3HB) production (grams per liter); and line plots, GFP fluorescence pBphaRsPP-gfp (phaR along with 232 bp-upstream region, PphaP),
(fluorescent unit/g-residual cell mass [FU/g-cells]). a H16/pBPP-gfp and f PHB-4/pBphaRsPP-gfp

because vegetable oils were potent candidates as resources fluorescence in C. necator H16 harboring various gfp-
for production of PHA copolymers by recombinant C. expression vectors, along with averages of cell growth
necator (Fukui et al. 1998; Mifune et al. 2008; Mifune et (RCM) and P(3HB) production (black and gray bars,
al. 2010; Tsuge et al. 2007). Figure 5a represents GFP
Appl Microbiol Biotechnol (2011) 89:1527– 15
respectively). RCMs reached a maximum (approx. 0.7
g/l)
 15 Appl Microbiol Biotechnol (2011) 89:1527–

3000
3.0 3000 3.0

2500
2.5 2500 2.5

(g/L) P(3HB)
Residual cell mass
2000
2.0 2000 2.0
GFP (FU/g-

(g/L) P(3HB)
Residual cell mass
1500
1.5 1500 1.5

GFP (FU/g-
1000
1.0 1000 1.0

500
0.5 500 0.5

0 0.0 0 0.0
0 20 40 60 80 10 20 30 40 50 60 70 80

Time (h) Time (h)

Fig. 5 Time-dependent changes of gfp expression by lac, tac, phaC,

open squares, H16/pBTac-gfp (Ptac); diamonds with dot, H16/pBPP-
and phaP promoters in recombinant strains of C. necator H16 grown
gfp (PphaP); and diamonds with cross hair; H16/pBphaRsPP-gfp
on 1.0% soybean oil. Black bars, residual cell mass (grams per liter);
(phaR along with 232 bp-upstream region, PphaP). b H16/pBBad-gfp
gray bars, P(3HB) production (grams per liter); and line plots, GFP
(PBAD) with addition of L-arabinose at 12 h. Open circles, no addition
fluorescence (fluorescent unit/g-residual cell mass [FU/g-cells]). a
of L- arabinose; open triangles, 0.01% L-arabinose; and open squares,
Open circles, H16/pBPC-gfp (PphaC); open triangles, H16/pBLac-gfp
0.1%
(Plac);
L-arabinose

The gfp expression was again strictly dependent on the

at 24 h similar to that observed in cultivation on fructose, addition of L-arabinose, while the level of GFP fluorescence
while P(3HB) production continued until 48 h and reached was much lower than those in the cells grown on fructose.
to approx. 2.2 g/l, which was 1.7-folds higher than P(3HB)
produced by the fructose-grown cells. As seen on fructose,
Ptac (H16/pBTac-gfp) was also strongest among the
promoters examined; the GFP fluorescence was highest at
the growth phase (>2,000 U/g-cells) and gradually
decreased during the PHA-biosynthesis phase. Plac (H16/
pBLac-gfp) and PphaC (H16/pBPC-gfp) mediated constitu-
tive gene expression also on soybean oil, leading the nearly
constant level of GFP fluorescence within 600–1,000
FU/g- cells. PphaP (H16/pBPP-gfp) was a slightly stronger
promoter than Plac and PphaC, although the gfp expression
at the growth phase was lower than those at the following
PHA-biosynthesis phase. The GFP fluorescence in H16/
pBphaRsPP-gfp was increased with increasing P(3HB)
accumulation and reached to maximum (669 FU/g-cells)
at 48 h. The expression of gfp by phaR-PphaP was also
tightly coupled with P(3HB) biosynthesis because GFP
fluorescence was negligible when PHB-4 harboring
pBphaRsPP-gfp or pBphaRlPP-gfp were cultivated on
soybean oil (data not shown). As seen on fructose, C.
necator cells harboring pBphaRlPP-gfp showed lower
growth and P(3HB) production rates than those harboring
pBPP-gfp or pBphaRsPP-gfp (data not shown).
H16/pBBad-gfp grown on soybean oil had a tendency to
show reduced P(3HB) production rates regardless of the
presence or absence of L-arabinose, as seen on fructose.
 Appl Microbiol Biotechnol (2011) 89:1527– 15
The maximum fluorescence with 0.1% L-arabinose was
1,070 FU/g-cells, and the gfp expression with 0.01% L-
arabinose was only present in faint levels throughout the
cultivation.

Discussion

Despite the potential of C. necator H16 as a host for

metabolic engineering aiming efficient microbial produc-
tion of PHAs, there has been only limited knowledge
regarding promoters for gene expression in this bacterium.
We consider that a collection of promoters by which gene
expression profiles are well evaluated is useful to achieve
effective modification of PHA biosynthesis and the
related carbon and energy metabolisms in C. necator. This
study investigated expression of gfp reporter gene in C.
necator under the control of five kinds of promoters.
Plac, Ptac, and PBAD have been well studied and often
applied in E. coli for inducible expression of genes
derived from various sources. The results in Fig. 3a
clearly demonstrated that in C. necator, Plac acted as a
promoter allowing constitutive gene expression. Ptac
mediated higher expression than Plac, although the
expression level gradu- ally decreased during the
stationary growth phase. Addition of IPTG gave no
effects on the gene expression by Plac alone, of which
result was consistent with the absence of a gene encoding
LacI homolog in C. necator genome. Interestingly,
H16/pBlacITac-gfp did not show detectable level of GFP
fluorescence throughout the cultivation even after the
addition of IPTG. This result indicated that LacI
 15
regulator expressed from the plasmid-borne gene certainly
repressed transcription by Ptac, and IPTG added into the
medium could not trigger derepression of transcription. It
was plausible that this would be caused by the lack of
ability for C. necator to incorporate β-galactosides includ-
ing IPTG, because any gene encoding a protein homolo-
gous to β-galactoside permease LacY was not found in C.
necator genome (Pohlmann et al. 2006).
Our previous studies have demonstrated that araC-PBAD
expression system was also functional in C. necator (Fukui
et al. 2002; Fukui et al. 2009), but the detail of the
expression profile has not been determined. The results
obtained here clearly indicated that the expression under
the control of araC-PBAD was strictly dependent on L-
arabinose concentration. On the other hand, we observed
slight decrease of cell growth and P(3HB) production in
the cells harboring pBBad-gfp. It was supposed that the
growth inhibition was caused by L-arabinose added into the
medium as an inducer, because RCMs at 24 h were slightly
decreased with increasing L-arabinose concentration (data
not shown). In contrast, the decrease of P(3HB) production
rates was irrespective to the addition of inducer, as
indicated by the decreased P(3HB) production on fructose
at 36 h with all the inducer concentrations examined
(Table 2). Since P(3HB) biosynthesis was not affected by
introduction of the other pBBR1-based vectors, the inhibi-
tion of P(3HB) biosynthesis was presumably due to the
heterologous expression of araC in C. necator. It should be
noted that the gfp expression gradually increased at a
constant rate throughout the cultivation, suggesting sus-
tained transcriptional activity of PBAD. This was probably
because L-arabinose, incorporated into the cells by
unknown transporter, was present at a constant intracellular
concentration due to lack of L-arabinose-assimilating
pathway in C. necator (Pohlmann et al. 2006). Despite
the slight negative effects on cell growth and P(3HB)
accumulation, the araC-PBAD system was useful for tightly
regulated gene expression in C. necator.
The expression vector pBPP-gfp harboring PphaP alone
mediated moderately high expression of gfp, and the
expression was independent on P(3HB) biosynthesis, most
likely due to insufficient amount of PhaR repressor within
the cells. Indeed, previous study has reported relatively
small amount of PhaR protein in C. necator (Pötter et al.
2002). The vectors containing phaR along with PphaP-gfp
fusion exhibited gfp expression tightly coupled with
P(3HB) accumulation as expected, although the
introduction of pBphaRlPP-gfp resulted in the decrease of
growth and P(3HB) production rates. The reason for this
phenomenon has been unclear yet, but we postulated that
the plasmid- borne phaR may be overexpressed by the long
upstream region of phaR on pBphaRlPP-gfp, and the
excess PhaR regulator may unexpectedly affect on global
gene expression
Appl Microbiol Biotechnol (2011) 89:1527–
leading to inhibition of cell growth and P(3HB)
biosynthesis. In contrast, the gfp expression without any
negative effects on the cellular functions was observed in
the strain harboring pBphaRsPP-gfp. Although the
expression by this vector was not so strong, further tuning
of PphaP region or construction of hybrid promoter would
allow us to obtain stronger promoter by which expression is
tightly coupled with PHA biosynthesis in C. necator. Such
autoregulatory expression of a target gene specific at the
PHA-biosynthesis phase would be applicable to engineer
carbon and energy metabolisms in C. necator for PHA
biosynthesis, such as enhancement of carbon flux toward
PHA after saturation of the cell growth.
Acknowledgements This study was supported by Industrial Tech-
nology Research Grant Program in 2005 from New Energy and
Industrial Technology Development Organization (NEDO),
KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas
Applied Genomics from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT).
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