Polymer Chemistry Laboratory and the RIKEN
Group of Japan Science and Technology Corporation, The Institute of
Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan
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INTRODUCTION |
Polyhydroxyalkanoates (PHAs) are
accumulated in various bacteria as intracellular carbon and energy
storage material under nutrient-limited conditions (3, 26,
30). These bacterial PHAs are expected to become attractive
alternatives for petrochemically based plastics, since they are
biodegradable thermoplastics. More than 90 different constituent
monomer units have been found (47). The PHA-producing
bacteria can be broadly divided into two groups according to the number
of carbon atoms in the monomeric units of the PHAs produced
(44). One group of bacteria, including Ralstonia
eutropha (formerly Alcaligenes eutrophus), produces short chain length PHAs with C3 to C5 monomer
units, while the other group, including Pseudomonas
oleovorans, produces medium chain length PHAs with C6
to C14 monomer units (3, 43).
Although the majority of bacteria accumulate either short chain length
PHA or medium chain length PHA, several bacteria have been found to
synthesize polyesters containing both short and medium chain length
3-hydroxyalkanoic acids (3HA). The bacteria Rhodospirillum
rubrum (4), Rhodocyclus gelatinosus
(27), and Rhodococcus sp. (13)
produced terpolyesters consisting of 3HA units of C4,
C5, and C6 from hexanoate. Aeromonas
caviae produced a random copolymer of 3-hydroxybutyrate (3HB) and
3-hydroxyhexanoate (3HHx) (6, 8, 38). Pseudomonas
strain GP4BH1 produced PHA containing 3HB and 3-hydroxyoctanoate (3HO)
from octanoate and PHA containing 3HB, 3HO, and 3-hydroxydecanoate
(3HD) from gluconate (46). In this bacterium, a polymer
blend was suggested to be synthesized rather than a copolymer. A
recombinant strain of P. oleovorans expressing R. eutropha poly(3HB) [P(3HB)] biosynthesis genes has been shown to
synthesize a blend of a P(3HB) homopolymer and a copolymer of 3HHx and
3HO units when grown on octanoate (49). Both polyesters were
stored as separated granules within the cells (32). In
addition, Pseudomonas fluorescens and several other
Pseudomonas strains were found to produce a
poly(3HB-co-3HA) [P(3HB-co-3HA)] copolymer
consisting of 3HA units of C4 to C12 from 3HB
and 1,3-butanediol (25). Although Thiocapsa
pfennigii accumulated only a P(3HB) homopolymer from various
carbon sources, a recombinant P. putida strain harboring the
PHA synthesis genes of T. pfennigii produced a
P(3HB-co-3HHx-co-3HO) terpolymer from octanoate
(28).
We have reported that Pseudomonas sp. strain 61-3 isolated
from soil produces a blend of a P(3HB) homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3HA units of
C4 to C12 from sugars and alkanoic acids
(1, 19, 20). In addition, two different types of polyester
granules were formed in the same cell (9, 21). This suggests
that Pseudomonas sp. strain 61-3 possesses two types of
polyester synthases with different substrate specificities, that is,
polyhydroxybutyrate (PHB) synthase and PHA synthase, specific for 3HB
and 3HA units ranging from C4 to C12,
respectively. In this study, we cloned and sequenced the P(3HB)
biosynthesis genes, as well as the P(3HB-co-3HA)
biosynthesis genes, of Pseudomonas sp. strain 61-3. The
substrate specificity of each polyester synthase was evaluated by
heterologous expression in PHA-negative mutants of P. putida
and R. eutropha. In addition, we found that the
phbRPs gene product exhibits significant
similarity to the AraC/XylS family of transcriptional activators and
report that it is a positive regulatory protein that controls the
expression of the P(3HB) biosynthesis operon.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
The
bacterial strains and plasmids used in this study are listed in Table
1, and the DNA fragments on vectors are
illustrated in Fig. 1B and D. Pseudomonas sp. strain 61-3 and P. putida and R. eutropha strains were cultivated at 30°C in a
nutrient-rich (NR) medium containing 10 g of meat extract, 10 g of Bacto Peptone (Difco), and 2 g of yeast extract (Difco) in 1 liter of distilled water. Escherichia coli strains were
grown at 37°C on Luria-Bertani (LB) medium (34). When
needed, kanamycin (50 mg/liter), tetracycline (12.5 mg/liter), or
ampicillin (50 mg/liter) was added to the medium.
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FIG. 1.
Organization of phb and pha loci
in Pseudomonas sp. strain 61-3 and DNA fragments including
the phb or pha locus on the broad-host-range
vector used in this study. (A) Restriction map of the 6.0-kb
HindIII-ApaI region and organization of
phbBPs, phbAPs,
phbCPs, and phbRPs. (C)
Restriction map of the 6.0-kb EcoRI-PstI region
and organization of ORF1, phaC1Ps,
phaZPs, phaC2Ps, and
phaDPs. (B and D) DNA fragments including the
phb and pha loci used in this study,
respectively. A, ApaI; B, BamHI; Bg,
BglII; E65, EcoO65I; H, HindIII;
Pv, PvuI; S, ScaI; Sc, SacI; Sp,
SpeI. Translational stop codons ( ) are present in
all three reading frames upstream from the GalK start codon to prevent
translational initiation elsewhere on the plasmid from traversing the
galK gene and interfering with translation initiation at the
galK start codon (22).
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Production and analysis of PHA.
Cells were cultivated on a
reciprocal shaker (130 strokes/min) at 30°C for 72 h in 500-ml
flasks containing 100 ml of a nitrogen-limited mineral salt (MS)
medium, which consisted of 0.9 g of
Na2HPO4 · 12H2O, 0.15 g
of KH2PO4, 0.05 g of NH4Cl,
0.02 g of MgSO4 · 7H2O, and 0.1 ml
of a trace element solution (19). Filter-sterilized carbon
sources were added to the medium as indicated in the text. Determination of cellular PHA content and composition by gas
chromatography, isolation of the accumulated PHA, fractionation of the
isolated polyesters with acetone, and nuclear magnetic resonance (NMR) analysis of polyesters were carried out as described by Kato et al.
(19, 20).
DNA manipulations.
Isolation of total genomic DNA and
plasmids, digestion of DNA with restriction endonucleases, agarose gel
electrophoresis, and transformation of E. coli were carried
out by standard procedures (34) or as recommended by the
manufacturers. DNA restriction fragments were isolated from agarose
gels by using a QIAEX II Gel Extraction Kit (QIAGEN). All other
DNA-manipulating enzymes were used as recommended by the manufacturers.
Genomic DNA libraries of Pseudomonas sp. strain 61-3 were
constructed with Charomid 9-28 (Nippon Gene) and pLA2917 (2)
by in vitro packaging using Gigapack II (Stratagene). Conjugation of
Pseudomonas sp. strain 61-3, P. putida, or
R. eutropha with E. coli S17-1 harboring
broad-host-range plasmids was performed as described by Friedrich et
al. (7).
Hybridization experiments.
Hybridization was carried out as
described by Southern (42). The DNA probes used were the PHB
synthase gene of R. eutropha (phbCRe)
(31, 36, 40) and a 24-mer synthetic oligonucleotide, 5'-CC(G/C)CAGATCAACAAGTT(C/T)TA(C/G)GAC-3', whose
sequence was based on that of a highly conserved region of the
polyester synthases of R. eutropha and P. oleovorans as described by Timm and Steinbüchel (50). Preparation of digoxigenin-labeled probes and
detection of hybridization signals on membranes were carried out with a DIG DNA Labeling and Detection Kit (Boehringer Mannheim) and a DIG
Oligonucleotide Tailing Kit (Boehringer Mannheim).
Nucleotide sequence analysis.
DNA fragments to be sequenced
were subcloned into pBluescript II KS+. DNA was sequenced
by the modified dideoxy-chain termination method basically as described
by Sanger et al. (35) with a 310 Genetic Analyzer (Perkin
Elmer). The sequencing reaction was performed in accordance with the
manual supplied with the dye terminator cycle sequencing kit (Perkin
Elmer). The resulting nucleotide sequence was analyzed with SDC-GENETYX
genetic information processing software (Software Development Co.,
Tokyo, Japan).
Plasmid construction.
Plasmids pJHS60, carrying
phbRPs and phbBACPs, and
pJHS48, carrying phbBACPs, were constructed by
introduction of the 6.0-kbp HindIII-ApaI
region and the 4.8-kbp ScaI-BamHI region into a
broad-host-range vector, pJRD215 (5), as
HindIII-SpeI fragments, respectively. pJHS60dBA containing phbRPs and
phbCPs was constructed by eliminating the
1.6-kbp PvuI fragment from pJHS60. pJASc22, pJASc60, and
pJASc60dC1Z were constructed as follows. The 2.2-kbp
EcoRI-XbaI region containing phaC1Ps and the 6.0-kbp
EcoRI-PstI region containing
phaC1ZC2DPs were introduced into pJRD215 as 2.2- and 6.0-kbp ApaI-SacI fragments to form pJASc22
and pJASc60, respectively. pJASc60dC1Z containing phaC2DPs was constructed by eliminating a
BglII-SphI region from a pBluescript II
KS+ derivative plasmid carrying the 6.0-kbp
EcoRI-PstI region and introducing the deleted
fragment into pJRD215 at the ApaI and SacI sites.
To analyze the effect of the PhbRPs protein on
transcriptional activity, plasmids pJZBB85R and pJZBB73 were
constructed as follows. The upstream region of the
phbBPs gene was inserted into the multiple
restriction site linker upstream from the promoterless galK'/lacZ gene in low-copy-number plasmid pFZY1
(22) at the HindIII site, with or without
phbRPs, and then inserted into pJRD215 at the
BamHI site as 8.5- and 7.3-kbp
BamHI-BglII fragments including galK'/lacZ, lacY, and lacA to obtain
pJZBB85R and pJZBB73, respectively (Fig. 1B). Translational stop codons
are present in all three reading frames between the 5' region of
phbBPs and the galK'/lacZ gene. This
eliminates the formation of unnecessary fusion protein, and GalK'/LacZ
hybrid protein, which displays LacZ activity but no GalK activity,
initiates at the ATG codon as described by Koop et al. (22).
-Galactosidase assay.
Recombinant Pseudomonas
sp. strain 61-3 grown in NR medium (NR condition) was collected by
centrifugation, washed twice with sterilized water, and then
transferred into MT medium (nutrient-limited condition) consisting of
1 g of NH4Cl, 2 g of NaCl, 1.4 g of
KH2PO4, 3.6 g of
Na2HPO4 · 12H2O, 0.02 g
of MgSO4 · 7H2O, 1 ml of a trace element
solution, and 15 g of glucose per 1 liter of 83 mM Tris-HCl buffer
(pH 7.2). The cells grown in NR or MT medium were disrupted with a
French press (96 MPa), and -galactosidase activity in the lysate was
determined basically by the method of Miller et al. (29), by
measuring the rate of increase in A420 resulting from the hydrolysis of
o-nitrophenyl--D-galactopyranoside to o-nitrophenol (ONP). The molar absorption coefficient of ONP
used to calculate the activity was 4.5 × 103
M
1 cm1. The activity was assayed in duplicate.
Disruption of phbCPs.
pBR322 was
used as an integration vector to inactivate the chromosomal
phbCPs gene of Pseudomonas sp. strain
61-3. A pBR322 derivative, designated pBREP9, was constructed by
subcloning the 941-bp EcoRI-PstI fragment of
phbCPs, which is a main part of phbCPs truncated at the 5' end (342 bp) and the
3' end (418 bp) with the NspI restriction site converted to
an EcoRI site, into pBR322 at the EcoRI and
PstI sites. Plasmid pBREP9 (Tcr) was then
introduced into Pseudomonas sp. strain 61-3 cells by electroporation with a Gene Pulser (Bio-Rad) as described by Iwasaki et
al. (17), with some modifications. Electrocompetent cells of
Pseudomonas sp. strain 61-3 were prepared in 8 mM HEPES
buffer (pH 7.2) containing 272 mM sucrose, and electroporation of the cells was performed with settings of 1.5 kV (7.5 kV/cm), 800 
, and
25 µF. The cells were grown in LB medium at 28°C for 12 h and,
after electroporation, incubated on LB agar medium containing 12.5-mg/liter tetracycline. To verify the integration into the chromosome in a single-crossover event via homologous recombination between the truncated phbCPs gene of pBREP9 and
the intact phbCPs gene on the chromosome,
Southern hybridization analysis (34) was carried out for
PvuII-digested genomic DNAs of 10 tetracycline-resistant clones. One strain
(phbCPs::tet), in which the
tetracycline resistance gene (tet) was integrated into
chromosomal phbCPs by homologous recombination,
was selected and used for further study.
Nucleotide sequence accession numbers.
The nucleotide
sequence data determined here will appear in the EMBL, GenBank, and
DDBJ databases under accession no. AB014757 and AB014758.
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RESULTS |
Cloning and identification of phb and pha
loci of Pseudomonas sp. strain 61-3.
To identify the
two possible types of polyester synthase genes in
Pseudomonas sp. strain 61-3, genomic DNA fragments from digestions with several restriction enzymes were hybridized with two
different gene probes. One probe is a 1.8-kbp fragment carrying the PHB
synthase gene of R. eutropha
(phbCRe), and the other is a 24-mer synthetic
oligonucleotide previously used for identification of PHA synthase
genes from pseudomonads (50). Southern hybridization analysis using each probe showed different patterns of strong signals
(14-kbp HindIII, 20-kbp EcoRI, 30-kbp
BamHI, 3.5-kbp PstI, and 6.3-kbp SacI
fragments with the phbCRe probe and 17-kbp
HindIII, 1.9-kbp EcoRI, 16-kbp
BamHI, 3.2-kbp PstI, and 19-kbp SacI
fragments with the 24-mer oligonucleotide probe). This suggested that
the two types of polyester synthase genes are located on different DNA
loci in Pseudomonas sp. strain 61-3.
For cloning of the polyester synthase gene hybridized with the
phbCRe and oligonucleotide probes, a genomic
sublibrary of 14-kbp HindIII fragments with cosmid
vector Charomid 9-28 and a total genomic DNA library with cosmid vector
pLA2917 (2) from partially digested genomic DNA using
Sau3AI were constructed by in vitro packaging. Positive
clones isolated by each hybridization screening were further analyzed
by Southern hybridization, and 6.0-kbp
HindIII-ApaI and 6.0-kbp
EcoRI-PstI regions were mapped as shown in Fig.
1A and C, respectively.
Organization of phb and pha loci.
The
complete nucleotide sequences of the cloned fragments were determined
in both strands. In the 6.0-kbp HindIII-ApaI
region (phb locus), four potential open reading frames
(ORFs) for protein-coding regions were identified by computer
analysis (Fig. 1A). The nucleotide sequence revealed homologies to
genes encoding PHB synthase (PhbCPs), -ketothiolase (PhbAPs), and NADPH-dependent
acetoacetyl coenzyme A (CoA) reductase (PhbBPs) in R. eutropha (Table 2). The
phb locus of Pseudomonas sp. strain 61-3 consisted of a phbBACPs operon, which is
different in organization from the corresponding operon in R. eutropha (phbCABRe).
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TABLE 2.
Homology of the products of the phb and
pha loci of Pseudomonas sp. strain 61-3 to
proteins of other bacteria
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In the region upstream of phbBPs, another ORF
(1,137 bp) was oriented in the direction opposite to that of the other
three genes (Fig. 1A). This ORF encoded a protein composed of 379 amino acids with a molecular mass of 42.3 kDa, and the deduced amino acid
sequence was similar to those of transcriptional regulator proteins
belonging to the AraC family, such as OruR of P. aeruginosa (25.7% identity, 67.6% similarity; Table 2) (14) and the
virulence-associated regulator of Mycobacterium tuberculosis
(24.8% identity, 61.4% similarity) (12). Accordingly, the
ORF was referred to as phbRPs. Several 
35 to
10 consensus sequences of 
70-dependent promoters were
found in the region between phbRPs and phbBPs on both strands by computer analysis
(Fig. 1A).
In the 6.0-kbp EcoRI-PstI region (pha
locus), there are several genes similar in organization to the
pha loci of P. oleovorans (16) and
P. aeruginosa (50). Two polyester synthase genes, referred to as phaC1Ps and
phaC2Ps, are represented as two large ORFs in
this region (Fig. 1C). The deduced amino acid sequences of both
phaC1Ps and phaC2Ps
exhibited greater identity to the PHA synthases of P. oleovorans (16) (Table 2) and P. aeruginosa (50) (54.7 to 83.7%) than to the PHB synthase of R. eutropha (31) (33.8 to 36.8%). The two PHA synthases
of Pseudomonas sp. strain 61-3 exhibited 53.2% identity to
each other, which is similar to the homology between the two synthases
of P. oleovorans (16). A putative PHA
depolymerase is encoded by phaZPs, which is
located between phaC1Ps and
phaC2Ps in Pseudomonas sp. strain
61-3. An ORF was also identified downstream of
phaC2Ps the deduced amino acid sequence of which
was similar to that of ORF3 of P. aeruginosa (Table 2)
(50); it was designated phaDPs, as
described by Steinbüchel et al. (45), although its
function is unknown. ORF1, upstream of phaC1Ps,
was similar to the 3'-terminal region of ORF2 of P. aeruginosa (81.7% identity for the C-terminal 93 amino acids) (50). Two nucleotide sequences resembling the 35 to 10
consensus sequence of the E. coli
70-dependent promoter and the 24 to 12 consensus
sequence of the E. coli 54-dependent promoter
were found upstream of phaC1Ps, although their relevance has not been explored.
Cys-301 of PhbCPs and Cys-296 of both PhaC1Ps
and PhaC2Ps in the lipase box-like sequence, which are
highly conserved in all known polyester synthases, are proposed to be
involved in the transesterification reaction, as well as Cys-319 in the
R. eutropha synthase (11).
Complementation studies and heterologous expression.
To
confirm whether the cloned fragments have functionally active PHA
biosynthesis genes, heterologous expression of the genes was
investigated in PHA-negative mutants P. putida GPp104
(16) and R. eutropha PHB4
(36). For expression of polyester synthase genes of
Pseudomonas sp. strain 61-3, pJHS60, pJHS60dBA, and pJHS48
harboring the PHB synthase gene and pJASc60, pJASc22, and pJASc60dC1Z
harboring the PHA synthase gene were constructed as described in
Materials and Methods. These plasmids were mobilized from E. coli S17-1 to P. putida GPp104 or R. eutropha PHB4. The transconjugants were cultivated
under nitrogen-limiting conditions in MS medium to promote PHA
biosynthesis from gluconate, octanoate, dodecanoate, or tetradecanoate
as a sole carbon source, and gas chromatography was used to determine
the content and composition of the accumulated PHA.
Tables 3 and
4 show the results of PHA accumulation in
the recombinant strains P. putida GPp104 and R. eutropha PHB4, respectively. Plasmids pJHS60,
pJHS60dBA, pJASc22, pJASc60, and pJASc60dC1Z could complement the
deficiency of polyester synthases in both of the mutant strains and
conferred the ability to accumulate PHA on the hosts. In contrast,
pJHS48 carrying phbBACPs without phbRPs was able to complement the mutation in
R. eutropha PHB4 but not in P. putida GPp104. PHB4/pJHS60 produced 30 to 47 wt%
(of dry cell weight) P(3HB) homopolymer from all of the carbon sources
examined (Table 4), and GPp104 harboring this plasmid also produced the
P(3HB) homopolymer from gluconate (20 wt%) and dodecanoate (1 wt%),
but not from octanoate (Table 3). However, GPp104/pJHS60dBA, in which
the phbBAPs gene had been deleted, accumulated
only a small amount (1 wt%) of the P(3HB) homopolymer from gluconate
(Table 3). Although P. putida GPp104 can supply medium chain
length (R)-3HA-CoA as a substrate for polyester synthases,
the recombinant strain harboring phbCPs produced
only the P(3HB) homopolymer. This indicates that PHB synthase of
Pseudomonas sp. strain 61-3 is specific for short chain
length (R)-3HA-CoA.
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TABLE 3.
Accumulation of PHA by recombinant P. putida
GPp104 harboring PHA biosynthesis genes of Pseudomonas sp.
strain 61-3a
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TABLE 4.
Accumulation of PHA by recombinant R. eutropha
PHB4 harboring PHA biosynthesis genes of
Pseudomonas sp. strain 61-3a
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The recombinant strains of GPp104 harboring pJASc60, pJASc22, and
pJASc60dC1Z produced the P(3HA) copolymer with C6 to
C12 monomer units from gluconate, and the main constituent
of the polyester was the 3HD unit (Table 3), whereas the 3HB unit was incorporated as a constituent of PHA in octanoate-, dodecanoate-, and
tetradecanoate-grown cells with pJASc22 and pJASc60, despite the low
content, ranging from 3 to 5 mol%. The compositions of the polyesters
that accumulated in the gluconate-grown cells carrying each of the
three plasmids were almost the same, while the fractions of 3HB and
3HHx in the alkanoate-grown cells were slightly higher with pJASc22
than with pJASc60dC1Z.
The strain PHB4 recombinants harboring pJASc60, pJASc22,
and pJASc60dC1Z produced P(3HB) homopolymer from gluconate, while they
produced a P(3HB-co-3HA) copolymer consisting of 3HA of
C4 to C12 monomer units from octanoate,
dodecanoate, or tetradecanoate with relatively high 3HB contents (Table
4). 3HB compositions of 30 to 50 mol% were incorporated into the
copolymers synthesized by the strains harboring pJASc22 and pJASc60dC1Z
from the alkanoates (Table 4). Interestingly,
PHB4/pJASc60 produced copolymers with a much larger 3HB
fraction (about 90 mol%) from octanoate and tetradecanoate. In order
to determine whether the polyesters synthesized by PHB4
carrying PHA synthase genes from alkanoates are random copolymers or
not, the parameter D values were calculated based on the sequence distribution of 3HB and 3HA units by 13C NMR analysis as
described by Kamiya et al. (18), which suggested that these
polyesters are random copolymers of 3HB and 3HA units (D values of 1.4 to 1.6). As a consequence, both PhaC1Ps and
PhaC2Ps of Pseudomonas sp. strain 61-3 were
found to be able to incorporate a wide compositional range of 3HA units
of C4 to C12 into the polyester.
PhbRPs is a member of the AraC/XylS family of
transcriptional activators.
The motif program Pfam,
developed by Sonnhammer et al. (41), was used to search
phbRPs for its defined amino acid sequence motifs. The search revealed that 86 residues at the carboxy terminus of
PhbRPs (residues 252 to 337) correspond to the AraC/XylS
family of bacterial regulatory helix-turn-helix proteins
(10). The deduced amino acid sequence of PhbRPs
displays a significant degree of similarity to the carboxy termini of
the AraC/XylS family of positive regulatory proteins (10).
The phbRPs gene product contains a
helix-turn-helix motif (residues 253 to 274) within the third quarter
of the polypeptide which is also similar to those of members of the
AraC/XylS family of transcriptional activators (10, 15, 33).
To examine the transcriptional activation of the promoter for the
phbBACPs operon by
phbRPs, transcriptional fusion genes were
constructed with or without phbRPs, as described
in Materials and Methods, to obtain pJZBB85R and pJZBB73, respectively
(Fig. 1B). -Galactosidase activities expressed in
Pseudomonas sp. strain 61-3 harboring these plasmids were
assayed (29). After the recombinants were grown in NR medium
at 30°C for 18 h, the cells were transferred to nitrogen-limited
MT medium and subsequently incubated for 4 h. As shown in Table
5, cells of Pseudomonas sp.
strain 61-3/pJZBB85R exhibited significantly higher -galactosidase
activity than those harboring pJZBB73 or control plasmid pJRD215 in
both of the media. This result indicates that there is a promoter in
the upstream region of phbBPs and that the
transcription from this promoter is activated by
phbRPs.
pJHS48 carrying phbBACPs without
phbRPs could not confer the ability to
accumulate PHA on P. putida GPp104 (Table 3), although it
complemented the mutation in R. eutropha PHB4
(Table 4). This means that PhbRPs is necessary for the
heterologous expression of phbBACPs in strain
GPp104 but not in strain PHB4. When pJSH18 carrying only
phbRPs without phbBACPs
was introduced into Pseudomonas sp. strain 61-3, the strain
not only produced a polyester with a higher cellular polyester content
(51 wt%) but also synthesized a polyester with a much larger 3HB
fraction (94 mol%) than the control strain harboring pJRD215 (17 wt%
and 44 mol% 3HB) from 1.5% glucose. On the basis of the fact that Pseudomonas sp. strain 61-3 accumulated a blend of P(3HB)
and P(3HB-co-3HA), amplification of
phbRPs seemed to enhance P(3HB) biosynthesis in
this bacterium, which resulted in the increase of the polyester content
and enrichment of the 3HB fraction in the whole polyester. These
results strongly suggest that PhbRPs is a transcriptional
activator for phbBACPs in Pseudomonas strains.
Isolation of a phbCPs-negative mutant of
Pseudomonas sp. strain 61-3.
The participation of PHB
synthase encoded by phbCPs in the PHA
biosynthesis by Pseudomonas sp. strain 61-3 was investigated by the gene disruption technique. A
phbCPs-negative form of Pseudomonas sp. strain 61-3, in which the tetracycline resistance gene
(tet) was integrated into chromosomal
phbCPs by homologous recombination (phbCPs::tet), was
successfully obtained by electroporation with pBREP9. Table
6 shows the PHA biosynthesis ability of
Pseudomonas sp. strain 61-3 (phbCPs::tet). The cells
were cultivated in MS medium containing glucose, and the accumulated
PHAs were isolated by chloroform and then fractionated with acetone.
While wild-type Pseudomonas sp. strain 61-3 accumulated an
acetone-soluble P(3HB-co-3HA) copolymer and an insoluble
P(3HB) homopolymer, the phbCPs-negative mutant
synthesized an acetone-soluble P(3HB-co-3HA) copolymer only,
and no polyester was recovered in the acetone-insoluble fraction.
Clearly, phbCPs-derived PHB synthase actually
synthesized a P(3HB) homopolymer in Pseudomonas sp. strain
61-3.
The P(3HB-co-3HA) copolymer synthesized by the
phbCPs::tet strain
contained 37 mol% of the 3HD unit as a main fraction and 25 mol% of
the 3HB unit. When plasmid pJSH18 carrying
phbRPs was introduced into the
phbCPs disruptant, the 3HB fraction of the copolymer was greatly increased to 61 mol%. 13C NMR
analysis revealed that the acetone-soluble polymer was a random
copolymer (D value of 1.3). Additional copies of
phbRPs were found to affect the content and
composition of the P(3HB-co-3HA) copolymer in
Pseudomonas sp. strain 61-3 cells.
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DISCUSSION |
Identification of phb and pha loci in
Pseudomonas sp. strain 61-3.
Previous studies have
suggested that Pseudomonas sp. strain 61-3 possesses two
types of polyester synthases with different substrate specificities
(19-21). In this study, the phb and
pha genes, which are located at different DNA loci in this
bacterium, were cloned and analyzed at the molecular level. The
nucleotide sequence of the phb locus revealed that P(3HB)
biosynthesis genes were constituted of the
phbBACPs operon. Furthermore,
phbRPs was also identified, and its
translational product was predicted to be a regulatory protein that
controls the transcription of the phbBACPs
operon. At the pha locus, the two PHA synthase-encoding genes (phaC1Ps and
phaC2Ps) flanking the PHA depolymerase gene (phaZPs) and two adjacent ORFs (ORF1 and
phaDPs) were identified.
Although P. putida provides medium chain length
(R)-3HA-CoA through de novo fatty acid synthesis and
-oxidation pathways from sugars and fatty acids, heterologous
expression of phbCPs in the PHA-negative mutant
P. putida GPp104 resulted in the accumulation of a P(3HB)
homopolymer, and medium chain length 3HA units from any of the carbon
sources examined were never detected. Furthermore, a
phbCPs-negative form of
Pseudomonas sp. strain 61-3, phbCPs::tet, accumulated a
P(3HB-co-3HA) copolymer only. It has been concluded that the
P(3HB) fraction in a polymer blend produced by wild-type Pseudomonas sp. strain 61-3 is formed by the function of the
PHB synthase encoded by phbCPs. These facts
indicated that PhbCPs is active for short chain length
(R)-3HA-CoA only. In contrast, introduction of
phaC1Ps and phaC2Ps into
P. putida GPp104 restored the ability to synthesize a P(3HA)
copolymer with C6 to C12 monomer units from
gluconate, and the 3HB unit could be detected in the copolymer produced
from alkanoates by the transconjugants harboring phaC1Ps, although the fraction was as small as 3 to 5 mol%. R. eutropha PHB4 harboring
phaC1Ps and/or phaC2Ps
produced P(3HB-co-3HA) copolymers consisting of 3HA units of
4 to 12 carbons from alkanoates. These results indicate that both PHA
synthases of Pseudomonas sp. strain 61-3 are able to
incorporate the 3HB unit into the polyester, as well as medium chain
length 3HA units.
The reason why the 3HB unit was detected at a low level in the
polyester produced by the transconjugants of strain GPp104 may be the
inefficiency with which (R)-3HB-CoA is provided through the
fatty acid metabolic pathways in this host. GPp104/pJHS60dBA, in which
the phbBAPs genes were deleted from pJHS60,
accumulated much less of a P(3HB) homopolymer (1 wt%) from gluconate
than when it harbored pJHS60 intact (20 wt%). This suggests that
(R)-3HB-CoA molecules are insufficiently supplied from
gluconate in P(3HA)-producing P. putida and that the
introduction of phbBAPs into strain GPp104 restored the pathway for (R)-3HB-CoA formation via
dimerization of acetyl-CoA and reduction of acetoacetyl-CoA catalyzed
by PhbAPs and PhbBPs, respectively. The 3HB
unit composition of the polyesters produced by PHB4
transconjugants was much higher than that of the polyesters produced by
GPp104 transconjugants, owing to the existence of an efficient pathway
by which to provide (R)-3HB-CoA via the dimerization of
acetyl-CoA in R. eutropha PHB4
transconjugants. Accumulation of the P(3HB) homopolymer from gluconate
in R. eutropha PHB4 harboring
phaC1Ps and/or phaC2Ps
suggests a defect in the key enzyme converting intermediates of de novo
fatty acid synthesis to medium chain length (R)-3HA-CoA as
substrates for the heterologous PHA synthase in R. eutropha cells.
From the results of heterologous expression described above, the
composition of the polyester accumulated was proved to be affected by
the substrate-supplying system in the host cells, as well as the
substrate specificities of the polyester synthases. It has been
reported that 17 to 26 mol% of the 3HB unit can be incorporated into
the octanoate-derived polyester accumulated in R. eutropha
PHB4 harboring the PHA synthase genes of P. aeruginosa, although P. aeruginosa produces medium
chain length PHA (50). Furthermore, Kraak et al.
(23) have reported that PHA synthase 1 of P. oleovorans shows relatively high
(R)-3-hydroxyvaleryl-CoA activity in vitro, despite the
incorporation of only a small fraction of the 3HV unit (less than 3 mol%) into PHA from odd-numbered substrates in vivo. They have
mentioned that PHA synthesis in vitro might allow an even greater range
of monomers to be incorporated than via in vivo PHA accumulation. The
substrate-supplying pathway for PHA synthases in host cells is
important for control of the monomer composition of PHA.
PhaC1Ps and PhaC2Ps exhibited similar substrate
specificities and were capable of incorporating a wide range of 3HA
units into PHA. In alkanoate-grown recombinants of GPp104,
PhaC1Ps was considered to have a tendency to incorporate
3HB and 3HHx into the polyester rather than PhaC2Ps, while
the 3HB fraction in the polyester synthesized by recombinant strains
PHB4 was higher with phaC2Ps than
with phaC1Ps. The alkanoate-derived polyester
content of PHB4/pJASc22 was higher than that of
gluconate-derived polyester, whereas the opposite was true of
PHB4/pJASc60dC1Z. The reason for this difference remains
unclear, but it might be the difference between the expression levels
of phaC1Ps and phaC2Ps,
depending on the host strains. Additional copies of the synthase gene
in the wild-type P. oleovorans and P. putida
strains have been shown to affect the monomer composition of PHA,
resulting in more of the 3HHx unit and less of the 3HO and 3HD units
compared with those of the unaltered wild-type strains (24).
The levels and duration of phaC1Ps and
phaC2Ps expression in Pseudomonas
strains and R. eutropha need to be investigated. Another
possible reason is that the translational product of
phaDPs expressed together with
phaC2Ps might affect the PHA biosynthesis of
strain PHB4, although the function of PhaDPs
is unknown.
Function of PhbRPs.
phbRPs,
located upstream of phbBPs and in the opposite
direction, encoded a protein exhibiting significant similarity to the AraC/XylS family of transcriptional activators (10, 15, 33). pJHS48 carrying phbBACPs without
phbRPs hardly conferred the ability to
accumulate PHA on P. putida GPp104. pJSH18 carrying only
phbRPs resulted in a drastic increase in the
content and enrichment of the 3HB fraction of the polyesters in
Pseudomonas sp. strain 61-3. From these results,
phbRPs was suggested to be necessary for P(3HB) biosynthesis in Pseudomonas strains and actually elevated
levels of -galactosidase activity in cells carrying the
phb promoter with lacZYA. That is,
PhbRPs is certainly an activator of the transcription of
phbBACPs under control of the phb
promoter. The existence of a putative regulatory protein in the PHA
biosynthesis operon is reported here for the first time. In contrast to
these phenomena seen in Pseudomonas strains, no difference
in the polyester contents synthesized by R. eutropha
PHB4 carrying phbBACPs with or
without phbRPs was recognized, indicating that
PhbRPs was not essential for the expression of
phbBACPs genes in strain PHB4. The
transcription of phbBACPs is likely to be
independent of phbRPs in strain
PHB4. In practice, several putative promoters were found
in the region upstream of phbBPs by computer
analysis, and phbPs genes might be transcribed
in strain PHB4 by one of these promoters.
Amplification of phbRPs in the
phbC::tet mutant of
Pseudomonas sp. strain 61-3 resulted in an increase in the
3HB unit in the P(3HB-co-3HA) copolymer from 19 to 53 mol%.
The excess PhbRPs molecules may have promoted a high level
of transcription of phbBAPs in the
phbC::tet strain, and more
(R)-3HB-CoA molecules may have been converted from
acetyl-CoA by PhbBAPs in the cells. Such efficient formation of (R)-3HB-CoA may promote incorporation of the
3HB unit into the P(3HB-co-3HA) copolymer by the function of
PHA synthases encoded by phaC1Ps and
phaC2Ps, resulting in enrichment of the 3HB
fraction in the accumulated copolymer. The results described here
demonstrate that metabolic modification of the PHA biosynthesis pathway
in Pseudomonas sp. strain 61-3 makes it possible to
synthesize the P(3HB-co-3HA) random copolymer with a novel composition.
We are indebted to H. G. Schlegel
(Georg-August-Universtät) for the kind gifts of E. coli S17-1 and R. eutropha PHB4 and to B. Witholt (ETH) for those of P. putida GPp104 and plasmid pJRD215 used in this work.
This work was supported by CREST (Core Research for Evolutional Science
and Technology) of Japan Science and Technology Corporation (JST).
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Abstract
Introduction
