Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes in Pseudomonas sp. Strain 61-3

Abstract

Two types of polyhydroxyalkanoate (PHA) biosynthesis gene loci (phb and pha) of Pseudomonas sp. strain 61-3, which produces a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer {poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA]} consisting of 3HA units of 4 to 12 carbon atoms, were cloned and analyzed at the molecular level. In the phb locus, three open reading frames encoding polyhydroxybutyrate (PHB) synthase (PhbC_Ps), β-ketothiolase (PhbA_Ps), and NADPH-dependent acetoacetyl coenzyme A reductase (PhbB_Ps) were found. The genetic organization showed a putative promoter region, followed by phbB_Ps-phbA_Ps-phbC_Ps. Upstream from phbB_Ps was found the phbR_Ps gene, which exhibits significant similarity to members of the AraC/XylS family of transcriptional activators. The phbR_Ps gene was found to be transcribed in the opposite direction from the three structural genes. Cloning of phbR_Ps in a relatively high-copy vector in Pseudomonas sp. strain 61-3 elevated the levels of β-galactosidase activity from a transcriptional phb promoter-lacZ fusion and also enhanced the 3HB fraction in the polyesters synthesized by this strain, suggesting that PhbR_Ps is a positive regulatory protein controlling the transcription of phbBAC_Ps in this bacterium. In the pha locus, two genes encoding PHA synthases (PhaC1_Ps and PhaC2_Ps) were flanked by a PHA depolymerase gene (phaZ_Ps), and two adjacent open reading frames (ORF1 and phaD_Ps), and the gene order was ORF1, phaC1_Ps, phaZ_Ps, phaC2_Ps, and phaD_Ps. Heterologous expression of the cloned fragments in PHA-negative mutants of Pseudomonas putida and Ralstonia eutropha revealed that PHB synthase and two PHA synthases of Pseudomonas sp. strain 61-3 were specific for short chain length and both short and medium chain length 3HA units, respectively.

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 phbR_Ps 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.

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.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristics	Source or reference(s)a
Strains
Pseudomonas sp. strain 61-3	Wild type	JCM 10015
		1, 19, 20
Pseudomonas sp. strain 61-3(phbC::tet)	Inactivation of chromosomal phbCPs by integration of Tcr; phbCPs-negative mutant	This study
P. putida GPp104	PHA-negative mutant of KT2442	16
R. eutropha PHB−4	PHA-negative mutant of H16	DSM 541; 36
E. coli DH5α	deoR endA1 gyrA96 hsdR17 (rK− mK+) relA1 supE thi-1 Δ(lacZYA-argFV169) φ80ΔlacZΔM15F−λ−	Clontech
E. coli S17-1	recA and tra genes of plasmid RP4 integrated into chromosome; auxotrophic for proline and thiamine	39
Plasmids
Charomid 9-28	Cosmid; Apr	Nippon Gene
pLA2917	Cosmid; Kmr Tcr RK2 replicon; Mob+	2
pJRD215	Cosmid; Kmr Smr RSF1010 replicon; Mob+	5
pBluescript II KS+	AprlacPOZ T7 and T3 promoter	Stratagene
pBR322	Apr Tcr	48
pFZY1	Apr F′ replicon; lacZYA	22
pJHS60	pJRD215 derivative; phbRPsphbBPsphbAPsphbCPs	This study
pJHS60dBA	pJRD215 derivative; phbRPsphbCPs	This study
pJHS48	pJRD215 derivative; phbBPsphbAPsphbCPs	This study
pJSH18	pJRD215 derivative; phbRPs	This study
pJASc22	pJRD215 derivative; phaC1Ps	This study
pJASc60	pJRD215 derivative; phaC1PsphaZPsphaC2PsphaDPs	This study
pJASc60dC1Z	pJRD215 derivative; phaC2PsphaDPs	This study
pJZBB85R	pJRD215 derivative; phbRPs; phb promoter, lacZYA	This study
pJZBB73	pJRD215 derivative; phb promoter; lacZYA	This study
pBREP9	0.9-kb NspI-PstI fragment of phbCPs in pBR322; Tcr	This study

^aJCM, Japan Collection of Microorganisms; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH. 

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 phbB_Ps, phbA_Ps, phbC_Ps, and phbR_Ps. (C) Restriction map of the 6.0-kb EcoRI-PstI region and organization of ORF1, phaC1_Ps, phaZ_Ps, phaC2_Ps, and phaD_Ps. (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).

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 (phbC_Re) (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 phbR_Ps and phbBAC_Ps, and pJHS48, carrying phbBAC_Ps, 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 phbR_Ps and phbC_Ps 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 phaC1_Ps and the 6.0-kbp EcoRI-PstI region containing phaC1ZC2D_Ps were introduced into pJRD215 as 2.2- and 6.0-kbp ApaI-SacI fragments to form pJASc22 and pJASc60, respectively. pJASc60dC1Z containing phaC2D_Ps 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 PhbR_Ps protein on transcriptional activity, plasmids pJZBB85R and pJZBB73 were constructed as follows. The upstream region of the phbB_Ps 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 phbR_Ps, 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 phbB_Ps 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).

β-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 β-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-β-d-galactopyranoside to o-nitrophenol (ONP). The molar absorption coefficient of ONP used to calculate the activity was 4.5 × 10³ M⁻¹ cm⁻¹. The activity was assayed in duplicate.

Disruption of phbC_Ps.

pBR322 was used as an integration vector to inactivate the chromosomal phbC_Ps gene of Pseudomonas sp. strain 61-3. A pBR322 derivative, designated pBREP9, was constructed by subcloning the 941-bp EcoRI-PstI fragment of phbC_Ps, which is a main part of phbC_Ps 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 (Tc^r) 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 Ω, 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 phbC_Ps gene of pBREP9 and the intact phbC_Ps gene on the chromosome, Southern hybridization analysis (34) was carried out for PvuII-digested genomic DNAs of 10 tetracycline-resistant clones. One strain (phbC_Ps::tet), in which the tetracycline resistance gene (tet) was integrated into chromosomal phbC_Ps 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.

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 (phbC_Re), 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 phbC_Re 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 phbC_Re 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 (PhbC_Ps), β-ketothiolase (PhbA_Ps), and NADPH-dependent acetoacetyl coenzyme A (CoA) reductase (PhbB_Ps) in R. eutropha (Table 2). The phb locus of Pseudomonas sp. strain 61-3 consisted of a phbBAC_Ps operon, which is different in organization from the corresponding operon in R. eutropha (phbCAB_Re).

TABLE 2.

Homology of the products of the phb and pha loci of Pseudomonas sp. strain 61-3 to proteins of other bacteria

Gene product designation	Size of putative gene product (kDa)	Homology to other gene products
		Designationa	Amino acid identity (%)
PhbRPs	42.3	OruR	25.7
PhbBPs	26.7	PhbBRe	66.1
PhbAPs	40.6	PhbARe	65.8
PhbCPs	64.3	PhbCRe	53.0
PhaC1Ps	62.3	PhaC1Po	83.7
PhaZPs	31.7	PhaZPo	89.0
PhaC2Ps	62.8	PhaC2Po	74.8
PhaDPs	23.5	PhaDPa	77.2

^aOruR, transcriptional regulator for ornithine metabolism of P. aeruginosa (14); PhbB_Re, PhbA_Re, and PhbC_Re, NADPH-dependent acetoacetyl-CoA reductase, β-ketothiolase, and PHB synthase of R. eutropha, respectively (31, 37, 40); PhaC1_Po, PhaZ_Po, and PhaC2_Po, PHA synthase 1, PHA depolymerase, and PHA synthase 2 of P. oleovorans, respectively (16); PhaD_Pa, ORF3 of P. aeruginosa (50). 

In the region upstream of phbB_Ps, 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 phbR_Ps. Several −35 to −10 consensus sequences of ς⁷⁰-dependent promoters were found in the region between phbR_Ps and phbB_Ps 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 phaC1_Ps and phaC2_Ps, are represented as two large ORFs in this region (Fig. 1C). The deduced amino acid sequences of both phaC1_Ps and phaC2_Ps 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 phaZ_Ps, which is located between phaC1_Ps and phaC2_Ps in Pseudomonas sp. strain 61-3. An ORF was also identified downstream of phaC2_Ps the deduced amino acid sequence of which was similar to that of ORF3 of P. aeruginosa (Table 2) (50); it was designated phaD_Ps, as described by Steinbüchel et al. (45), although its function is unknown. ORF1, upstream of phaC1_Ps, 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 ς⁷⁰-dependent promoter and the −24 to −12 consensus sequence of the E. coli ς⁵⁴-dependent promoter were found upstream of phaC1_Ps, although their relevance has not been explored.

Cys-301 of PhbC_Ps and Cys-296 of both PhaC1_Ps and PhaC2_Ps 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 PHB⁻4 (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 PHB⁻4. 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 PHB⁻4, 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 phbBAC_Ps without phbR_Ps was able to complement the mutation in R. eutropha PHB⁻4 but not in P. putida GPp104. PHB⁻4/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 phbBA_Ps 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 phbC_Ps 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.

TABLE 3.

Accumulation of PHA by recombinant P. putida GPp104 harboring PHA biosynthesis genes of Pseudomonas sp. strain 61-3^a

Plasmid (relevant markers)	Substrate	Dry cell wt (g/liter)	PHA content (wt%)	PHA composition (mol%)b
				3HB (C4)	3HHx (C6)	3HO (C8)	3HD (C10)	3HDD (C12)	3H5DD (C12′)
pJHS60 (phbR phbB phbA phbC)	Gluconate	0.89	20	100	0	0	0	0	0
	Octanoate	0.87	Trace	100	0	0	0	0	0
	Dodecanoate	0.77	1	100	0	0	0	0	0
pJHS60dBA (phbR phbC)	Gluconate	0.80	1	100	0	0	0	0	0
pJHS48 (phbB phbA phbC)	Gluconate	0.80	0
pJASc60 (phaC1 phaZ phaC2 phaD)	Gluconate	0.62	6	0	1	8	53	23	15
	Octanoate	1.06	29	3	15	81	1	0	0
	Dodecanoate	0.96	21	5	13	37	29	16	0
	Tetradecanoate	0.89	23	4	11	37	31	17	0
pJASc22 (phaC1)	Gluconate	0.88	9	0	2	15	58	14	11
	Octanoate	1.17	43	3	16	77	2	0	0
	Dodecanoate	0.61	33	3	12	39	29	17	0
	Tetradecanoate	0.97	38	3	12	39	28	18	0
pJASc60dC1Z (phaC2 phaD)	Gluconate	0.95	10	0	1	13	62	14	10
	Octanoate	0.84	6	0	10	72	18	0	0
	Dodecanoate	0.64	5	0	7	39	29	25	0
	Tetradecanoate	0.61	5	0	6	33	34	27	0

^aCells were cultivated at 30°C for 72 h in MS medium containing the sodium salt of gluconate (2% wt/vol), octanoate, dodecanoate, or tetradecanoate (0.5% wt/vol) as the sole carbon source. 

^b3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecenoate. 

TABLE 4.

Accumulation of PHA by recombinant R. eutropha PHB⁻4 harboring PHA biosynthesis genes of Pseudomonas sp. strain 61-3^a

Plasmid (relevant markers)	Substrate	Dry cell wt (g/liter)	PHA content (wt%)	PHA composition (mol%)b
				3HB (C4)	3HHx (C6)	3HO (C8)	3HD (C10)	3HDD (C12)	3H5DD (C12′)
pJHS60 (phbR phbB phbA phbC)	Gluconate	1.62	47	100	0	0	0	0	0
	Octanoate	1.06	40	100	0	0	0	0	0
	Dodecanoate	0.72	30	100	0	0	0	0	0
pJHS60dBA (phbR phbC)	Gluconate	1.56	51	100	0	0	0	0	0
	Octanoate	1.09	35	100	0	0	0	0	0
	Dodecanoate	0.61	26	100	0	0	0	0	0
pJHS48 (phbB phbA phbC)	Gluconate	1.71	54	100	0	0	0	0	0
	Octanoate	1.75	33	100	0	0	0	0	0
	Dodecanoate	0.51	14	100	0	0	0	0	0
pJASc60 (phaC1 phaZ phaC2 phaD)	Gluconate	0.85	12	100	0	0	0	0	0
	Octanoate	0.84	6	92	0	8	0	0	0
	Dodecanoate	0.30	6	100	0	0	0	0	0
	Tetradecanoate	0.69	4	91	0	3	3	3	0
pJASc22 (phaC1)	Gluconate	0.74	2	100	0	0	0	0	0
	Octanoate	0.86	13	31	10	59	0	0	0
	Dodecanoate	0.61	5	31	4	23	23	19	0
	Tetradecanoate	0.97	14	46	4	21	18	11	0
pJASc60dC1Z (phaC2 phaD)	Gluconate	0.92	20	100	0	0	0	0	0
	Octanoate	0.74	4	50	7	43	0	0	0
	Dodecanoate	0.34	1	51	0	9	13	27	0
	Tetradecanoate	0.67	5	44	1	16	15	24	0

^aCells were cultivated at 30°C for 72 h in MS medium containing the sodium salt of gluconate (2% wt/vol), octanoate (0.1% [wt/vol] × 5), dodecanoate, or tetradecanoate (0.5% wt/vol) as the sole carbon source. 

^b3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecenoate. 

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 PHB⁻4 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, PHB⁻4/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 PHB⁻4 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 PhaC1_Ps and PhaC2_Ps 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.

PhbR_Ps 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 phbR_Ps for its defined amino acid sequence motifs. The search revealed that 86 residues at the carboxy terminus of PhbR_Ps (residues 252 to 337) correspond to the AraC/XylS family of bacterial regulatory helix-turn-helix proteins (10). The deduced amino acid sequence of PhbR_Ps displays a significant degree of similarity to the carboxy termini of the AraC/XylS family of positive regulatory proteins (10). The phbR_Ps 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 phbBAC_Ps operon by phbR_Ps, transcriptional fusion genes were constructed with or without phbR_Ps, as described in Materials and Methods, to obtain pJZBB85R and pJZBB73, respectively (Fig. 1B). β-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 β-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 phbB_Ps and that the transcription from this promoter is activated by phbR_Ps.

TABLE 5.

β-Galactosidase levels expressed in Pseudomonas sp. strain 61-3 harboring pJZBB85R or pJZBB73

Plasmid (relevant markers)	β-Galactosidase activity (U/mg of protein)a
	NR mediumb (1st stage)		MT mediumc (2nd stage)
	12 h	18 h	4 h
pJRD215 (none)	6	NDd	ND
pJZBB85R (phbRPsphb promoter lacZYA)	46	64	123
pJZBB73 (phb promoter lacZYA)	5	5	23

^aOne unit corresponds to the formation of 1 nmol of ONP per min at 28°C. The activity was assayed in duplicate. 

^bCells were cultivated in NR medium at 30°C. 

^cCells grown in NR medium at 30°C for 18 h were transferred to MT medium and subsequently incubated at 30°C for 4 h. 

^dND, not determined. 

pJHS48 carrying phbBAC_Ps without phbR_Ps could not confer the ability to accumulate PHA on P. putida GPp104 (Table 3), although it complemented the mutation in R. eutropha PHB⁻4 (Table 4). This means that PhbR_Ps is necessary for the heterologous expression of phbBAC_Ps in strain GPp104 but not in strain PHB⁻4. When pJSH18 carrying only phbR_Ps without phbBAC_Ps 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 phbR_Ps 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 PhbR_Ps is a transcriptional activator for phbBAC_Ps in Pseudomonas strains.

Isolation of a phbC_Ps-negative mutant of Pseudomonas sp. strain 61-3.

The participation of PHB synthase encoded by phbC_Ps in the PHA biosynthesis by Pseudomonas sp. strain 61-3 was investigated by the gene disruption technique. A phbC_Ps-negative form of Pseudomonas sp. strain 61-3, in which the tetracycline resistance gene (tet) was integrated into chromosomal phbC_Ps by homologous recombination (phbC_Ps::tet), was successfully obtained by electroporation with pBREP9. Table 6 shows the PHA biosynthesis ability of Pseudomonas sp. strain 61-3 (phbC_Ps::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 phbC_Ps-negative mutant synthesized an acetone-soluble P(3HB-co-3HA) copolymer only, and no polyester was recovered in the acetone-insoluble fraction. Clearly, phbC_Ps-derived PHB synthase actually synthesized a P(3HB) homopolymer in Pseudomonas sp. strain 61-3.

TABLE 6.

Acetone fractionation of PHA accumulated in Pseudomonas sp. strain 61-3 (phbC_Ps::Tet)^a

Strain	Plasmid (relevant marker)	Fraction	PHA yield (mg)	PHA composition (mol%)c
				3HB (C4)	3HHx (C6)	3HO (C8)	3HD (C10)	3HDD (C12)	3H5DD (C12′)
Pseudomonas sp. strain 61-3b		Whole	363	87	2	5	6	0	0
		Acetone soluble	262	44	5	21	25	2	3
		Acetone insoluble	101	100	0	0	0	0	0
Pseudomonas sp. strain 61-3 (phbC::tet)		Whole	101	27	0	13	35	12	13
		Acetone soluble	100	25	0	11	37	13	14
		Acetone insoluble	Trace
Pseudomonas sp. strain 61-3 (phbC::tet)	pJSH18 (phbRPs)	Whole	100	62	0	7	18	6	7
		Acetone soluble	96	61	0	7	19	6	7
		Acetone insoluble	4	94	0	0	6	0	0

^aCells were cultivated at 30°C for 48 h in MS medium containing 1.5% (wt/vol) glucose. 

^bKato et al. (19). 

^c3HDD, 3-hydroxydodecanoate; 3H5DD, 3-hydroxy-cis-5-dodecenoate. 

The P(3HB-co-3HA) copolymer synthesized by the phbC_Ps::tet strain contained 37 mol% of the 3HD unit as a main fraction and 25 mol% of the 3HB unit. When plasmid pJSH18 carrying phbR_Ps was introduced into the phbC_Ps 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 phbR_Ps were found to affect the content and composition of the P(3HB-co-3HA) copolymer in Pseudomonas sp. strain 61-3 cells.

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 phbBAC_Ps operon. Furthermore, phbR_Ps was also identified, and its translational product was predicted to be a regulatory protein that controls the transcription of the phbBAC_Ps operon. At the pha locus, the two PHA synthase-encoding genes (phaC1_Ps and phaC2_Ps) flanking the PHA depolymerase gene (phaZ_Ps) and two adjacent ORFs (ORF1 and phaD_Ps) were identified.

Although P. putida provides medium chain length (R)-3HA-CoA through de novo fatty acid synthesis and β-oxidation pathways from sugars and fatty acids, heterologous expression of phbC_Ps 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 phbC_Ps-negative form of Pseudomonas sp. strain 61-3, phbC_Ps::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 phbC_Ps. These facts indicated that PhbC_Ps is active for short chain length (R)-3HA-CoA only. In contrast, introduction of phaC1_Ps and phaC2_Ps 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 phaC1_Ps, although the fraction was as small as 3 to 5 mol%. R. eutropha PHB⁻4 harboring phaC1_Ps and/or phaC2_Ps 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 phbBA_Ps 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 phbBA_Ps into strain GPp104 restored the pathway for (R)-3HB-CoA formation via dimerization of acetyl-CoA and reduction of acetoacetyl-CoA catalyzed by PhbA_Ps and PhbB_Ps, respectively. The 3HB unit composition of the polyesters produced by PHB⁻4 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 PHB⁻4 transconjugants. Accumulation of the P(3HB) homopolymer from gluconate in R. eutropha PHB⁻4 harboring phaC1_Ps and/or phaC2_Ps 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 PHB⁻4 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.

PhaC1_Ps and PhaC2_Ps exhibited similar substrate specificities and were capable of incorporating a wide range of 3HA units into PHA. In alkanoate-grown recombinants of GPp104, PhaC1_Ps was considered to have a tendency to incorporate 3HB and 3HHx into the polyester rather than PhaC2_Ps, while the 3HB fraction in the polyester synthesized by recombinant strains PHB⁻4 was higher with phaC2_Ps than with phaC1_Ps. The alkanoate-derived polyester content of PHB⁻4/pJASc22 was higher than that of gluconate-derived polyester, whereas the opposite was true of PHB⁻4/pJASc60dC1Z. The reason for this difference remains unclear, but it might be the difference between the expression levels of phaC1_Ps and phaC2_Ps, 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 phaC1_Ps and phaC2_Ps expression in Pseudomonas strains and R. eutropha need to be investigated. Another possible reason is that the translational product of phaD_Ps expressed together with phaC2_Ps might affect the PHA biosynthesis of strain PHB⁻4, although the function of PhaD_Ps is unknown.

Function of PhbR_Ps.

phbR_Ps, located upstream of phbB_Ps and in the opposite direction, encoded a protein exhibiting significant similarity to the AraC/XylS family of transcriptional activators (10, 15, 33). pJHS48 carrying phbBAC_Ps without phbR_Ps hardly conferred the ability to accumulate PHA on P. putida GPp104. pJSH18 carrying only phbR_Ps 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, phbR_Ps was suggested to be necessary for P(3HB) biosynthesis in Pseudomonas strains and actually elevated levels of β-galactosidase activity in cells carrying the phb promoter with lacZYA. That is, PhbR_Ps is certainly an activator of the transcription of phbBAC_Ps 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 PHB⁻4 carrying phbBAC_Ps with or without phbR_Ps was recognized, indicating that PhbR_Ps was not essential for the expression of phbBAC_Ps genes in strain PHB⁻4. The transcription of phbBAC_Ps is likely to be independent of phbR_Ps in strain PHB⁻4. In practice, several putative promoters were found in the region upstream of phbB_Ps by computer analysis, and phb_Ps genes might be transcribed in strain PHB⁻4 by one of these promoters.

Amplification of phbR_Ps 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 PhbR_Ps molecules may have promoted a high level of transcription of phbBA_Ps in the phbC::tet strain, and more (R)-3HB-CoA molecules may have been converted from acetyl-CoA by PhbBA_Ps 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 phaC1_Ps and phaC2_Ps, 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.