INTRODUCTION

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Polyhydroxyalkanoates (PHA) occur naturally in a wide variety of bacteria as a material for storage of carbon and energy from renewable carbon resources under conditions of restricted growth. They have recently attracted industrial attention because of their potential properties as biodegradable thermoplastics. A great deal of research has been undertaken with the aim of understanding the mechanism of PHA biosynthesis (1, 7), and numerous advances have been made from recent molecular analysis of PHA biosynthesis genes (29, 30).

In Alcaligenes eutrophus, two molecules of acetyl coenzymeA (acetyl-CoA) derived from various carbon sources are converted to (R)-3-hydroxybutyryl-CoA [(R)-(3HB)-CoA] via dimerization catalyzed by -ketothiolase and subsequent (R)-specific reduction catalyzed by NADPH-acetoacetyl-CoA reductase. The resultant (R)-3HB-CoA molecules are then polymerized into poly(3-hydroxybutyrate) [P(3HB)] by the function of PHA synthase. The genes encoding the three enzymes are organized in a single operon as phbCAB, consisting of the genes for PHA synthase, thiolase, and reductase, respectively (21, 25, 28). This operon has been introduced into several prokaryotes and eukaryotes, resulting in P(3HB) accumulation (18, 22, 29, 34). Pseudomonads belonging to rRNA homology group I accumulated PHA consisting of medium-chain-length (C₅ to C₁₄) 3-hydroxyalkanoate (3HA) units from simple carbon sources, such as sugars (13, 15, 32) or n-alkanoic acids, n-alkanols, or n-alkanes (4, 12, 14, 16). In the production of PHA from alkanoic acids with six to nine carbon atoms by Pseudomonas oleovorans, the major monomer unit in PHA has the same chain length as the alkanoic acids fed as a carbon source, but monomer units with more or fewer carbon atoms than the acid fed are also generally present in the PHA formed. It has been proposed that acyl-CoA intermediates of the -oxidation pathway are channeled to PHA biosynthesis (6a, 9); however, few details have been elucidated.

Aeromonas caviae isolated from soil has been reported to produce a random copolyester of 3HB and (R)-3-hydroxyhexanoate [(R)-3HHx], P(3HB-co-3HHx), from alkanoic acids with even carbon numbers or from plant oils (8, 26). On the basis of the fact that this bacterium does not accumulate any PHA from sugars, C₄ and C₆ (R)-3HA units were proposed to be supplied from the -oxidation intermediates, similar to the PHA biosynthesis pathway in pseudomonads on alkanoic acids (8). Recently, we have cloned and analyzed the PHA biosynthesis genes of A. caviae and have suggested that ORF3 located downstream of the PHA synthase gene (phaC_Ac) encodes (R)-specific enoyl-CoA hydratase (11). In this paper, we report direct evidence that ORF3 is essential for PHA biosynthesis from alkanoic acids in A. caviae. Furthermore, ORF3, referred to as phaJ_Ac, is overexpressed in Escherichia coli, and characteristics of the translated product are investigated.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1. A. caviae strains were cultivated at 30°C in a nutrient-rich medium containing 10 g of meat extract, 10 g of polypeptone, and 2 g of yeast extract in 1 liter of distilled water, and E. coli strains were grown at 37°C on Luria-Bertani (LB) medium (23). Kanamycin (50 mg/liter) or ampicillin (50 mg/liter) was added to the medium when necessary.

Chemical mutagenesis and isolation of a PHA-negative mutant of A. caviae. A. caviae FA440 was treated with N-methyl-N'-nitro-N-nitrosoguanidine (50 µg/ml) and then inoculated on MM agar medium composed of 3 g of K₂HPO₄, 7 g of KH₂PO₄, 1 g of (NH₄)₂SO₄, 0.1 g of yeast extract, 5 g of glucose, and 15 g of agar in 1 liter of distilled water (pH 7.0). Colonies grown at 30°C for 5 days were replicated on MPA agar medium composed of 7 g of K₂HPO₄, 3 g of KH₂PO₄, 1 g of (NH₄)₂SO₄, 0.1 g of MgSO₄ · 7H₂O, 0.1 g of yeast extract, 1.5 g of palmitic acid, 10 ml of Triton X-100, and 15 g of agar in 1 liter of distilled water (pH 7.0). The small amount of yeast extract (0.1 g/liter) was added to enhance cell growth. PHA-negative mutants, which were not stained with Sudan Black B (24) on MPA agar medium, were isolated.

DNA manipulation. Basic recombinant DNA techniques, such as preparation and purification of plasmid DNA, restriction endonuclease digestion, agarose gel electrophoresis, and transformation of E. coli, were carried out as described by Sambrook et al. (23). Transconjugation of A. caviae with E. coli S17-1 harboring broad-host-range plasmids was performed as described by Friedrich et al. (10).

Construction of pJRDG13. Two BglII sites across phaC_Ac were created on pEE32 (11) by site-directed mutagenesis with the unique-site elimination procedure (6) using mutagenic primers M2 (5'-GACGCTACGGGCTAGATCTCGCCTCGGGTGTG-3') and M5 (5'-GCGGCTCAACCCAGATCTTGCCTGCCCAACAG-3'), which corresponded to the sequences from nucleotides 2647 to 2678 and 4430 to 4461, respectively (11) (the created BglII sites are underlined). The resultant plasmid was digested with BglII and self-ligated to delete the coding region of phaC_Ac. The EcoRI restriction fragment excised from the phaC_Ac-deleted plasmid was inserted into pJRD215 (5) to form pJRDG13, harboring ORF1 and phaJ_Ac (ORF3) of A. caviae.

Production and analysis of PHA. A. caviae strains were first cultivated in 100 ml of nutrient-rich medium on a reciprocal shaker (130 strokes/min) at 30°C for 12 h. Then harvested and washed cells were transferred into 100 ml of nitrogen-free mineral salt medium (pH 7.0), which was composed of 0.9 g of Na₂HPO₄ · 12H₂O, 0.15 g of KH₂PO₄, 0.02 g of MgSO₄ · 7H₂O, and 0.1 ml of trace element solution (15), and incubated at 30°C for 48 h. Sodium dodecanoate (1%) was added as a carbon substrate for PHA biosynthesis. For maintenance of broad-host-range plasmids in A. caviae, kanamycin was added to the medium at a concentration of 50 mg/liter. Cellular PHA contents were determined by gas chromatography after methanolysis of dried cells in the presence of 15% sulfuric acid, as described previously (15).

Construction of expression plasmid pETNB3. To construct a plasmid for the overexpression of phaJ_Ac, an NdeI site was introduced into the proposed translational start codon of phaJ_Ac by PCR. The 427-bp fragment was amplified with primers P3N (5'-GCCATATGAGCGCACAATCCCTGGAAGTAG-3') and P3C (5'-CTGGGATCCGCCGGTGCTTAAGGCAGCTTG-3'), corresponding to the sequences from nucleotides 4470 to 4499 and 4867 to 4896 (complementary sequence), respectively (11) (underlined sequences show an NdeI site in P3N and a BamHI site in P3C). The PCR product was purified, digested with NdeI and BamHI, and subcloned into pET-3a. The resultant plasmid was termed pETNB3.

Enzyme assay. Enoyl-CoA hydratase activity was assayed by the hydration of crotonyl-CoA (19, 31). A 10-µl volume of enzyme solution was added to 290 µl of 50 mM Tris-HCl (pH 8.0) containing 0.25 mM crotonyl-CoA (Sigma) in quartz cuvettes with a 0.1-cm light path, and the decrease in absorbance at 263 nm was measured at 30°C. The ₂₆₃ of the enoyl-thioester bond is taken to be 6.7 × 10³ M¹ cm¹ (31). Other C₅, C₆, and C₈ trans-2-enoyl-CoA substrates used for the hydratase assay were synthesized from a lithium salt of CoA and the corresponding trans-2-alkenoic acids (Tokyo Kasei) based on a mixed-anhydride method and were purified with a Sep-Pak C₁₈ column (Waters), as described by Valentin and Steinbüchel (33). Protein concentrations were determined by the method of Bradford (2) by using Bio-Rad assay solution and bovine serum albumin as the standard.

Expression of phaJ_Ac in E. coli. The expression plasmid pETNB3 was transformed into E. coli BL21(DE3). A 1-ml overnight culture of the cells was inoculated into 100 ml of LB medium containing 100 mg of ampicillin per liter and cultivated at 30°C. Isopropyl--D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM when the absorbance at 600 nm reached 0.6, and cultivation was continued for an additional 2 h at 30°C. The cells grown in four 100-ml cultures were harvested, sonicated in buffer A (20 mM HEPES, pH 7.2), and subjected to centrifugation (20,000 × g, 30 min, 4°C). The obtained soluble protein fraction was loaded directly onto a HiLoad Q-Sepharose HP 16/10 column (Pharmacia) equilibrated with buffer A and was eluted with a linear gradient (250 ml) of 0 to 1.0 M NaCl (2.5 ml/min). The enoyl-CoA hydratase assay was done for each fraction (5 ml) with crotonyl-CoA used as a substrate, and the active fractions were combined, concentrated by ultrafiltration with a Centriplus 10 (Amicon), and desalted with a Sephadex G-25 column (Pharmacia). The purified enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (17). The N-terminal amino acid sequence of the enzyme was determined with a protein sequencer (model 610A; Perkin-Elmer) as instructed by the manufacturer.

Determination of molecular mass. The molecular mass of the native enzyme was evaluated by gel filtration chromatography with a Superdex 75 HR 10/30 column (Pharmacia) in buffer A containing 0.15 M NaCl. The molecular mass of the subunit was determined by SDS-PAGE and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS).

	RESULTS

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Complementation analysis of A. caviae PHA-negative mutants. Chemical mutagenesis was carried out to generate PHA-negative mutants of A. caviae FA440, and five mutants incapable of accumulating PHA on an MPA agar plate containing palmitic acid were isolated after 10⁴ colonies were screened. One such mutant, A. caviae AC004, was used for further analysis. The wild-type strain of A. caviae produced P(3HB-co-3HHx), up to 29 wt% of the dry cell weight from dodecanoate as a carbon substrate by two-step fermentation, whereas mutant AC004 could not synthesize any PHA under the same condition. As shown in Fig. 1b, the PHA phenotype of AC004 was able to revert to PHA⁺ by introduction of pJRDEE32, harboring a 3.2-kbp fragment of A. caviae genomic DNA (ORF1, phaC_Ac, and ORF3) (11), indicating that AC004 contains a mutation within the 3.2-kbp region in its chromosome. Various deletion clones of pJRDEE32 were then introduced into AC004, and the ability of the recombinant strains to synthesize PHA was investigated to identify the mutation. The transconjugants of AC004 harboring pJRDEE32d3 or pJRDEE32d13 did not accumulate any PHA or accumulated only a trace amount. In contrast, both pJRDEE32d1 and pJRDG13, harboring ORF3, could complement the PHA-negative mutation of AC004. These results have revealed that AC004 does not lack the PHA synthase encoded by phaC_Ac but contains a mutation within the ORF3 region. This is clear evidence that ORF3 (phaJ_Ac) is essential for PHA biosynthesis on alkanoic acids by A. caviae.

View larger version (21K): [in this window] [in a new window]   FIG. 1.   (a) Schematic drawing of a 3.2-kbp EcoRI fragment containing ORF1, phaCAc, and phaJAc (ORF3) with a putative promoter region from A. caviae FA440. (b) The ability of PJRDEE32 and its deleted clones to complement a PHA-negative mutant of A. caviae (AC004). PHA production was carried out on 1% dodecanoate by two-step fermentation, as described in the text.

Expression of phaJ_Ac in E. coli. When phaJ_Ac (ORF3) was expressed in E. coli DH5 together with phaC_Ac under the control of the native promoter, a higher enoyl-CoA hydratase activity was detected in the supernatant of the recombinant strain than in the control strain (11), suggesting that phaJ_Ac is a gene encoding enoyl-CoA hydratase. For further investigation of the translated product of phaJ_Ac, we constructed an expression plasmid, pETNB3, in which phaJ_Ac is oriented in the T7 promoter and designed ribosome binding site of pET-3a (Fig. 2). E. coli BL21(DE3) was then transformed with pETNB3 and cultured until mid-log phase at 30°C. After the addition of 0.4 mM IPTG and further cultivation for 2 h, a very high enoyl-CoA hydratase activity (1.7 × 10³ U/mg) was detected in the soluble protein fraction without formation of an inclusion body. This activity was more than 10³-fold higher than that in strain DH5 carrying phaC_Ac and phaJ_Ac under the control of the native promoter region. A protein of 15.5 kDa, which is in reasonable agreement with the approximate mass of the predicted phaJ_Ac product (14.1 kDa), was observed in the soluble fraction from BL21(DE3)/pETNB3 as determined by SDS-PAGE analysis (Fig. 3, lane 2).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Construction of plasmid pETNB3 for overexpression of phaJAc in E. coli BL21(DE3). A designed ribosome binding site (RBS) from pET-3a is indicated (underlined). PT7, T7 promoter in pET-3a.

View larger version (46K): [in this window] [in a new window]   FIG. 3.   SDS-PAGE analysis of enoyl-CoA hydratase from E. coli BL21(DE3)/pETNB3. Lanes: 1, molecular mass standard proteins, with the masses indicated on the left (from top to bottom, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, and soybean trypsin inhibitor); 2, crude extract proteins of E. coli BL21(DE3)/pETNB3; 3, purified enoyl-CoA hydratase after chromatography on Q-Sepharose.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Elution profile of enoyl-CoA hydratase from E. coli BL21(DE3)/pETNB3. The soluble protein fraction from the cells grown in four 100-ml cultures was applied to a Q-Sepharose column. , absorbance at 280 nm; , concentration of NaCl; , enoyl-CoA hydratase activity toward crotonyl-CoA.

Characteristics of the phaJ_Ac product. The molecular mass of the purified enoyl-CoA hydratase was determined as 13,963 Da by MALDI-TOFMS analysis, and the obtained value was in good agreement with a value (13,954 Da) calculated from the deduced amino acid sequence in which the initial Met was ignored. The N-terminal Met was deleted probably due to posttranslational modification in E. coli cells. The native molecular mass of the hydratase was estimated as 31,000 Da by gel filtration chromatography, indicating the formation of a homodimer.

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Evaluation of stereospecificity of enoyl-CoA hydratase encoded by phaJAc. (a) Hydration of crotonyl-CoA coupled with (S)-specific dehydrogenation of 3HB-CoA catalyzed by (S)-3HA-CoA dehydrogenase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 0.5 mM NAD+, 6 mU of (S)-3HA-CoA dehydrogenase, and 1 U of hydratase in 400 µl of 50 mM Tris-HCl (pH 8.0). (b) Hydration of crotonyl-CoA coupled with (R)-specific polymerization of 3HB-CoA catalyzed by crude PHA synthase. The reaction mixture was composed of 0.25 mM crotonyl-CoA, 10 mM DTNB, 1.2 mU of PHA synthase (a crude extract of A. eutrophus H16 containing 10 µg of protein), and 1 U of hydratase in 400 µl of 125 mM potassium phosphate (pH 7.2). The crude extract of A. eutrophus H16 was prepared from cells grown on fructose for 30 h at 30°C, as described previously (25). Symbols: , addition of phaJAc-derived enoyl-CoA hydratase; , addition of crotonase [(S)-specific enoyl-CoA hydratase]; , no addition of enoyl-CoA hydratase.

	DISCUSSION

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As the biosynthetic route to (R)-3HA-CoA from -oxidation intermediates for PHA biosynthesis, three candidates have been proposed: (R)-specific hydration of 2-enoyl-CoA, (R)-specific reduction of 3-ketoacyl-CoA, or epimerization of (S)-3HA-CoA (6a, 9, 29). The results described here clearly indicate that (R)-specific enoyl-CoA hydratase encoded by phaJ_Ac (ORF3) is involved in A. caviae PHA biosynthesis on alkanoic acids. The 2-enoyl-CoA intermediates with four to six carbon atoms derived from the -oxidation of longer alkanoic acids can be converted to corresponding (R)-3HA-CoA by the hydratase. The resultant C₄ to C₆ (R)-3HA-CoA molecules are acceptable as substrates for PHA synthase of A. caviae encoded by phaC_Ac, and then they are polymerized to P(3HB-co-3HHx), as shown in Fig. 6. This is the first study to prove that the (R)-specific hydration of 2-enoyl-CoA is a channeling pathway for supplying (R)-3HA-CoA monomer units for PHA biosynthesis through the fatty acid -oxidation pathway. The location of the gene encoding the (R)-hydratase within the pha locus is also a new finding.

View larger version (26K): [in this window] [in a new window]   FIG. 6.   Proposed pathway of P(3HB-co-3HHx) biosynthesis by A. caviae from alkanoic acids or oils. 1, -ketothiolase; 2, NADH-acetoacetyl-CoA dehydrogenase; 3, crotonase [(S)-specific enoyl-CoA hydratase].

Rhodospirillum rubrum is known to synthesize PHA consisting of short- and medium-chain-length 3HA (C₄ to C₆), like A. caviae (3), and the presence of two stereospecific [(R)- and (S)-specific] enoyl-CoA hydratases in R. rubrum has been reported previously (19). As to their function in P(3HB) biosynthesis from acetate, it has been proposed that the (R)-3HB unit is supplied from two acetyl-CoA molecules via four-step reactions catalyzed by -ketothiolase, NADH-acetoacetyl-CoA dehydrogenase, crotonase [(S)-specific enoyl-CoA hydratase], and (R)-specific enoyl-CoA hydratase. This pathway may function to supply the (R)-3HB unit from alkanoic acids in A. caviae together with the channeling route from the -oxidation (Fig. 6), since a small fraction (3 to 5 mol%) of the 3HB unit was incorporated into copolyesters synthesized from alkanoic acids with odd numbers of carbon atoms (8). The activity toward 2-hexenoyl-CoA of the (R)-specific enoyl-CoA hydratase from R. rubrum was approximately one-third of the activity toward crotonyl-CoA (19), which is similar to the property of the enzyme encoded by phaJ_Ac (Table 3). The (R)-specific hydratase of R. rubrum may also play an important role in PHA biosynthesis on alkanoic acids. However, further comparison of the properties of the (R)-specific hydratases of A. caviae and R. rubrum could not be made because of only a partial purification of the enzyme of R. rubrum. Methylobacterium rhodesianum, which is able to grow and to synthesize P(3HB) from methanol, has also been reported to possess two distinct crotonyl-CoA hydratases (20); however, the properties of the purified (R)-specific enzyme from M. rhodesianum (sigmoidal kinetics and a relatively high K_m) are quite different from those of the phaJ_Ac-derived hydratase. The participation of the (R)-hydratase in P(3HB) biosynthesis through the serine pathway in M. rhodesianum is still unclear.

PHA biosynthesis genes of A. caviae have been introduced into a PHA-negative mutant of A. eutrophus, and the ability of the transconjugants to synthesize PHA has been investigated (11). In some cases, the deletion of phaJ_Ac resulted in an increase of the 3HHx fraction in P(3HB-co-3HHx) synthesized by the recombinant strains of A. eutrophus. This phenomenon was probably due to the higher hydration activity of the phaJ_Ac product toward crotonyl-CoA than 2-hexenoyl-CoA. Expression of phaJ_Ac in host cells may result in a higher concentration of (R)-3HB-CoA than of (R)-3HHx-CoA, which is reflected in the lower 3HHx unit content in the PHA produced. This result may suggest the possibility of changing the 3HHx fraction in bacterial polyesters by controlled expression of phaJ_Ac.

The phaJ_Ac product has been suggested to have another function in the accumulation of PHA granules in cells, and ORF1 of A. caviae located upstream of phaC_Ac may also take part in PHA biosynthesis and accumulation (11). Further study will be done to elucidate the characteristics and functions of the genes surrounding phaC_Ac.