Characterization of 13 kDa Granule-Associated Protein in Aeromonas

Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan; and Department of Biological .... Applied Microbiology...
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Biomacromolecules 2001, 2, 148-153

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Characterization of 13 kDa Granule-Associated Protein in Aeromonas caviae and Biosynthesis of Polyhydroxyalkanoates with Altered Molar Composition by Recombinant Bacteria Toshiaki Fukui,†,‡ Tomoyasu Kichise,†,§ Tadahisa Iwata,† and Yoshiharu Doi*,†,§ Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan; and Department of Biological and Environmental Sciences, Saitama University, Shimo-Okubo, Urawa, Saitama 338-8570, Japan Received September 4, 2000

Analysis of native poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] inclusions from Aeromonas caViae FA440 revealed that ORF1 (a 348-bp gene located immediately upstream of phaCAc) encodes a 13-kDa granule-associated protein, which was referred to as phaPAc. Several recombinant strains of A. caViae were constructed and conducted to analyze their PHA-producing abilities. A transconjugant of FA440 harboring additional copies of phaPCJAc genes accumulated P(3HB-co-3HHx) copolyesters with much higher 3HHx composition (46-63 mol %) than wild-type strain from alkanoates or olive oil. Deletion analysis revealed that overexpression of phaJAc encoding monomer-supplying (R)-hydratase was not a reason for the compositional change in the recombinant strains. PHA synthase activity in PHA inclusion fraction from the transconjugant composed of 60 mol % of 3HHx was 10-fold higher than that from the strain FA440 with 13 mol % of 3HHx, suggesting an importance of the level of PHA synthase activity for controlling the PHA composition in vivo. Introduction Polyhydroxyalkanoates (PHA) are naturally occurring in a wide variety of bacteria as a carbon and energy storage material from renewable carbon resources under unbalanced growth conditions. They have recently attracted industrial attention because of their potential properties as biodegradable thermoplastics.1-3 A number of research have been made for understanding of the mechanism of PHA biosynthesis, and numerous advances have been obtained from the recent molecular analysis of PHA biosynthesis genes.4,5 PHA synthase, encoded by phbC or phaC, is a key enzyme of PHA biosynthesis which catalyzes the polymerization of (R)-3-hydroxyacyl-CoA monomers into water-insoluble macromolecules, and the reaction mechanism of this intriguing enzyme has been attracted and investigated.6-8 This enzyme is located on the surface of the intracellular PHA inclusions together with PHA depolymerase as active components. In addition to them, a third class of proteins without enzymatic activity is associated on the PHA inclusions in various PHAproducing bacteria.9,10 They are referred to as phasins9 and their structural genes, phaP, have been identified from Rhodococcus ruber,11 Chromatium Vinosum,12 Ralstonia eutropha (formerly Alcaligenes eutrophus),13,14 Acinetobacter sp.,15 Paracoccus denitorificans,16 Bacillus megaterium,17 and Pseudomonas strains.18,19 The importance of PhaP proteins * Corresponding author. Telephone: +81-48-467-9402. Fax: +81-48462-4667. E-mail: [email protected]. † RIKEN Institute. ‡ Present address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, YoshidaHonmachi, Sakyo-ku, Kyoto 606-8501, Japan. § Saitama University.

in PHA biosynthesis was indicated by the fact that a phaPnegative mutant of PHA producer accumulated less PHA than the wild-type strain and that the resultant PHA appeared to be a single large granule.13 Recently, a regulatory protein for pha locus has been proposed to be a class of proteins associated with PHA inclusions in Pseudomonas oleoVorans.18 So far, various recombinant bacteria have been constructed by introduction of multicopy number of pha genes and examined their PHA producing abilities3. In several studies, the contents of intracellular PHA were not elevated or only slightly elevated by amplification of phaC genes. We have investigated the PHA-producing abilities of phaC-negative mutant of R. eutropha complemented by PHA synthase gene of Aeromonas caViae, and demonstrated that the recombinant strains exhibiting quite different levels of synthase activity produced polyesters with a similar content and accumulation rate.20 It has been reported that the introduction of pha genes of P. oleoVorans into itself or Pseudomonas putida resulted in no increase of PHA content and a slight change in the composition.21,22 These phenomena suggest the multiple regulation of PHA biosynthesis pathways. Kraak et al. recently reported that PHA content in P. oleoVorans was increased significantly under non-nitrogen limiting conditions, when phaC1 was strongly expressed through Palk promoter.23 A. caViae was isolated as a producer of a random copolymer of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] from alkanoates or oils.24,25 We have cloned and analyzed the PHA biosynthesis genes of A. caViae including ORF1 with unknown function and structural

10.1021/bm0056052 CCC: $20.00 © 2001 American Chemical Society Published on Web 12/28/2000

Compositional Change of PHA in Recombinant Bacteria

genes of PHA synthase (phaCAc) and (R)-specific enoyl-CoA hydratase (phaJAc).26,27 Moreover, phaJAc has been proved to be an essential gene for supplying (R)-3HA-CoA monomers to PHA biosynthesis in A. caViae.27 In this study, a 13-kDa PHA-granule associated protein encoded by ORF1 was identified. Furthermore, we studied PHA-producing abilities of recombinant A. caViae and P. putida strains in which various sets of phaAc genes were introduced from plasmids. Materials and Methods Bacterial strains. A. caViae FA440,24,25 P. putida GPp104,28 and R. eutropha PHB-429 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. Escherichia coli DH5a and S17-130 were grown at 37 °C on a Luria-Bertani (LB) medium.31 Kanamycin (50 mg/L) or ampicillin (50 mg/L) was added to the medium, when necessary. Plasmids. A recombinant plasmid pJRDEE32 harboring PHA biosynthesis genes of A. caViae and its deleted plasmids (pJRDEE32d1, pJRDEE32d3, pJRDEE32d13, and pJRDG13) were reported previously.26,27 pJRDEE32d3A was constructed from pJRDEE32d3 by site-directed mutagenesis of the codon encoding Cys319 of PhaCAc to that of Ala under the unique site elimination methods32 using a mutagenic primer (5′CACGGCATCGGCTACGCCATCGGCGGCACCGCC-3′, the under line indicated the mismatches) and U.S.E. mutagenesis kit (Amersham Pharmacia). DNA manipulations were carried out according to Sambrook et al.31 Transconjugation of A. caViae, P. putida, and R. eutopha with E. coli S17-1 harboring broad-host-range plasmids was performed as described previously.26 Production and Analysis of PHA. PHA production was carried out on reciprocal shaker (130 strokes/min) at 30 °C for 72 h in 500 mL flasks with a 100 mL of nitrogen-limited mineral salts (MS) medium.33 The sodium salt of dodecanoate or tetradecanoate, or olive oil for A. caViae, or sodium salt of gluconate, hexanoate, or octanoate for P. putida was added in the medium as a sole carbon source, as indicated in the text. For maintenance of broad-host-range plasmids, kanamycin was added to the medium at a concentration of 50 mg/L. Cellular PHA content and composition were determined by gas chromatography after methanolysis of dried cells in the presence of 15% sulfuric acid, as described previously.33 TEM Analysis. Cells which had been washed and resupended in 100 mM potassium phosphate buffer (pH 6.8) were fixed in the buffer containing 2% glutaraledehyde, dehydrated by ethanol, and embedded in Epon812 resin. Ultrathin sections of the embedded cells on copper grids were doubly stained with uranyl acetate and lead(III) citrate. Micrographs were taken with a JEM-2000FXII electron microscopy operated at an acceleration voltage of 100 kV at room temperature. Isolation of Native PHA Granules. A. caViae cells grown in an MS medium containing dodecanoate (0.25% w/v) at 30 °C for 36 h were washed and resuspended in 100 mM

Biomacromolecules, Vol. 2, No. 1, 2001 149

Tris-HCl (pH 7.5). The cells were disrupted by a French press (96 Mpa) and then centrifuged (1000g, 5 min, 4 °C) to remove the cell debris and unbroken cells. The whole cell extract was loaded on the top of a discontinuous glycerol gradient. The gradient for granules from A. caViae FA440 and FA440/pJRDEE32 were prepared from 1 mL of 88% and 4 mL of 44% glycerol in 100 mM Tris-HCl and from 1 mL of 88%, 1 mL of 66%, and 2 mL of 22% glycerol in the buffer, respectively. After the ultracentrifugation (210000g, 30 min, 4 °C), granules at the interphase of 44-88% glycerol (for FA440) or that of 22-66% glycerol (for FA440/ pJRDEE32) were collected, following washing with the buffer. The granules resuspended in 1 mL of the buffer were subsequently loaded on a discontinuous sucrose gradient prepared from 1 mL each of 2.0, 1,67, 1.33, and 1.0 M sucrose in the buffer. After the ultracentrifugation (210000g, 2 h, 4 °C), a PHA granule layer was isolated, washed, and resuspended in the buffer. P(3HB-co-13 mol % 3HHx) granules produced by A. caViae FA440 were collected at the interphase of 1.33-1.67 M sucrose, and P(3HB-co-60 mol % 3HHx) by FA440/pJRDEE32 were at that of 0-1.0 M sucrose, respectively. Enzyme Assay. Whole cell extracts of A. caViae and P. putida grown on dodecanoate and octanoate, respectively, at 30 °C for 36-48 h were prepared as described above. PHA synthase activity in the cell extracts was determined by a spectroscopic assay using chemically synthesized (R)3HB-CoA and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and enoyl-CoA hydratase activity in soluble fractions of the whole extracts was assayed by the hydration of crotonylCoA (Sigma), as described previously.26-27 PHA synthase activity on the native granules was assayed toward (R)-3HB-CoA and (R)-3HHx-CoA generated in situ from the corresponding trans-2-enoyl-CoA with (R)-specific enoyl-CoA hydratase purified from E. coli BL21(DE3)/ pETNB3.27 The reaction mixture was composed of 0.25 mM of crotonyl-CoA or synthetic trans-2-hexenoyl-CoA, 1 U of (R)-enoyl-CoA hydratase, 10 mM DTNB in 400 µL of 50 mM potassium phosphate buffer (pH 7.2). After addition of granule suspension, the increase in absorbance at 412 nm was recorded at 30 °C. It has been confirmed that almost no absorbance change was observed when (R)-enoyl-CoA hydratase was omitted from the mixture. Electrophoresis. The cell extracts containing 10 µg of soluble proteins were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under the standard procedure. Western blot analysis of PhaCAc using the specific anti-serum toward the C-terminal oligopeptide of PhaCAc has been described previously.34 Protein concentrations were determined by using Bio-Rad assay solution and bovine serum albumin as the standard. Results Identification of a 13kDa Granule-Associated Protein and Its Structural Gene from A. caWiae. The native P(3HBco-3HHx) granules accumulated in A. caViae cells grown on dodecanoate were isolated by density-gradient ultracentrifugation. The PHA composition of the granule fraction

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Figure 1. SDS-PAGE (lanes 1, 2, 5, and 6) and Western-blot (lanes 3 and 4) analyses of cell extracts and native PHA granule fractions isolated from A. caviae grown on 0.25% w/v of sodium dodecanoate for 36 h at 30 °C. Lanes: 1 and 3, cell extract from FA440 containing 10 µg of soluble proteins; 2, 4, and 5, PHA granule fraction from FA440 containing 300 µg (lanes 2 and 4) or 200 µg (lane 5) of dry granules; 6, PHA granule fraction from FA440/pJRDEE32 containing 200 µg of dry granules.

Figure 2. Organization of PHA biosynthesis genes of A. caviae FA440.

was determined to be 87 mol % of 3HB and 13 mol % of 3HHx fractions by GC analysis, and proteins associated on surface of the inclusions were separated by SDS-PAGE, as shown in Figure 1, lane 2. The result clearly indicated that two proteins, 68 kDa (GA68) and 13 kDa (GA13) proteins, were mainly bound on the granules. The molecular mass of GA68 was consistent with the deduced mass of PHA synthase encoded by phaCAc (66.3 kDa), and Western-blot analysis using the specific antiserum34 indicated that GA68 was actually PHA synthase (lane 3). The N-terminal amino acid sequence of GA13 corresponded to the translated product of ORF1, which was located immediately upstream of phaCAc. This fact revealed that ORF1 was involved in PHA biosynthesis by encoding a granule-associated protein; therefore, this gene was designated as phaPAc. The PHA biosynthesis genes of A. caViae are organized as a phaPC-J cluster, as shown in Figure 2. A recombinant plasmid pJRDEE32 had been constructed by insertion of phaPCJAc with the native promoter region into a broad-host-range plasmid pJRD215, as reported previously.26 When a transconjugant of A. caViae FA440 harboring pJRDEE32 was cultivated on dodecanoate, a much greater amount of PhaPCAc proteins were bound on the PHA inclusions in comparison with the wild-type strain, as shown in Figure 1, lanes 5 and 6. In addition, transmission electron microscopic examination of the FA440/pJRDEE32 cells revealed the presence of numerous number of small granules in the cells (Figure 3B), while the granules accumulated in the strain FA440 were fewer and bigger (Figure 3A).

Fukui et al.

Figure 3. Electron micrographs of ultrathin sections of A. caviae FA440 (A) and of FA440/pJRDEE32 (B) grown on 0.25% of sodium dodecanoate at 30 °C for 36 h. The scale bars represent 0.5 mm.

PHA Production by Recombinant A. caWiae and P. putida. A. caViae FA440 has been reported to synthesize P(3HB-co-3HHx) copolyester from fatty acids longer than C10 or plant oils as a sole carbon source.24 As shown in Table 1, this bacterium accumulated the copolyester up to 22-36 wt % in dry cells from dodecanoate, tetradecanoate, and olive oil. The mol fractions of 3HHx unit were nearly constant at approximately 20 mol % in the copolyesters from various carbon sources. A transconjugant FA440/pJRDEE32 accumulated PHA with slightly larger content than FA440, while 3HHx fractions in the produced PHA reached to 4663 mol %. The introduction of additional copies of phaPCJAc genes resulted in a drastic change of copolyester composition. To clarify which gene took part in this compositional change, each plasmid carrying the deleted phaAc gene(s) (pJRDEE32d1, pJRDEE32d3, pJRDEE32d13, or pJRDG13)26,27 was transferred into the strain FA440, and PHA-producing abilities of these transconjugants were investigated. The strain harboring pJRDEE32d3 (phaJAc-deleted derivative) accumulated the copolyester with higher content (40-49 wt %) and higher 3HHx fraction (46-52 mol %) than wild-type strain, as well as FA440/pJRDEE32. In contrast, such composition change was not observed when phaPAc or phaCAc was deleted from the pha locus on the plasmid (pJRDEE32d1, pJRDEE32d13, or pJRDG13). These facts suggested that coexpression of phaPCAc genes caused the increase of 3HHx fraction in the copolyesters. PHA-negative mutant of P. putida, GPp104, was also examined as a heterologous host of pha genes from A. caViae, as also shown in Table 1. The transconjugants harboring phaCAc not only revert the phenotype of GPp104 to PHApositive but also confer the ability to synthesize P(3HB-co3HHx) copolyesters. Although PHA contents on gluconate were low in all transconjugants, their PHA-producing abilities on hexanoate and octanoate were different depending on the introduced plasmid. GPp104/pJRDEE32 (phaPCJAc) accumulated the largest amount of P(3HB-co-3HHx) with 3040 mol % of 3HHx from the alkanoates, as reported previously.26 The strain harboring phaPCAc genes (pJRDEE32d3) not only accumulated the copolyesters as well as that harboring pJRDEE32 but also synthesized copolyesters with quite high 3HHx fraction (71 mol %) from hexanoate. In contrast to these strains, GPp104 harboring pJRDEE32d1 or pJRDEE32d13, in which phaPAc had been deleted from the pha

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Compositional Change of PHA in Recombinant Bacteria Table 1. Accumulation of PHA in Recombinant Strains of A. caviae FA440 and P. putida GPp104a

strain

plasmid (relevant markers)b

carbon source

A. caviae none FA440

dodecanoate tetradecanoate olive oil pJRDEE32 dodecanoate (phaPCJAc) tetradecanoate olive oil pJRDEE32d1 dodecanoate (phaCJAc) tetradecanoate olive oil pJRDEE32d3 dodecanoate (phaPC Ac) tetradecanoate olive oil PJRDEE32d13 dodecanoate (phaCAc) tetradecanoate olive oil PJRDG13 dodecanoate (phaPJAc) tetradecanoate olive oil

P. putida none GPp104

gluconate hexanoate octanoate pJRDEE32 gluconate (phaPCJAc) hexanoate octanoate pJRDEE32d1 gluconate (phaCJAc) hexanoate octanoate pJRDEE32d3 gluconate (phaPCAc) hexanoate octanoate pJRDEE32d13 gluconate (phaCAc) hexanoate octanoate

dry PHA cell cont compn (mol %) weight (wt (g/L) %) 3HB 3HH 1.51 1.41 1.24 1.55 1.91 1.68 1.10 1.52 1.36 1.42 1.15 0.99 1.12 1.60 1.21 1.30 1.66 1.66

36 31 22 54 48 19 39 33 19 49 47 40 37 40 20 36 38 17

87 84 79 37 41 54 85 80 74 48 54 52 84 79 76 77 72 74

13 16 21 63 59 46 15 20 26 52 46 48 16 21 24 23 28 26

0.65 0.71 0.60 1.02 0.91 1.42 0.73 0.72 0.81 0.91 1.21 1.22 0.71 0.75 0.75

0 0 0 4 37 48 trace 2 5 4 37 37 trace 1 5

71 60 69 63 85 53 29 47 45 72

29 40 31 37 15 47 71 53 55 28

a Cells were cultivated for 72 h at 30 °C in an MS medium containing the sodium salt of dodecanote or tetradecanoate (0.5% w/v) or olive oil (1% w/v) for A. caviae strains or the sodium salt of gluconate (1.5% w/v), hexanoate, or octanoate (0.5% w/v) for P. putida strains.

operon, accumulated only small amount of polyesters even from the alkanoates. Likewise in A. caViae FA440, coexpression of phaCAc with phaPAc in P. putida GPp104 greatly affected the biosynthesis of P(3HB-co-3HHx) copolyesters from alkanoates. Enzyme Assay and Electrophoretic Analysis. We further carried out enzyme assay and electrophoretic analysis for each transconjugant. Table 2 shows the activities of PHA synthase and enoyl-CoA hydratase (including both (S)- and (R)-specific enzymes) in crude extracts prepared from the cells at early stationary phase. Both FA440/pJRDEE32 and FA440/pJRDEE32d3, which accumulated P(3HB-co-3HHx) with high 3HHx fractions (46-58 mol %), exhibited a 10fold higher PHA synthase activity (568 and 519 U/g protein, respectively) than FA440 (52 U/g protein). SDS-PAGE and Western-blot analyses clearly indicated that both PhaPCAc proteins were strongly expressed in these transconjugants (Figure 4A). On the contrary, PHA synthase activities and expression of phaCAc in the strains harboring pJRDEE32d1

Figure 4. (A) Expression of PhaCAc (immuno-staining) and PhaPAc (Coomassie Brilliant Blue-staining) in recombinant strains of A. caviae FA440. Lanes: 1 and 8, FA440; 2, FA440/pJRDEE32; 3, FA440/ pJRDEE32d1; 4, FA440/pJRDEE32d3; 5, FA440/ pJRDEE32d13; 6, FA440/pJRDG13; 7, FA440/pJRDEE32d3A. (B) Expression of PhaCAc (immuno-staining) in recombinant strains of P. putida GPp104. Lanes: 9, GPp104/pJRDEE32; 10, GPp104/ pJRDEE32d1; 11, GPp104/pJRDEE32d3; 12, GPp104/pJRDEE32d13. The extracts containing 10 µg of soluble proteins were prepared from the cells grown for 36 h at 30 °C on sodium dodecanoate (0.25% w/v) for A. caviae or sodium octanoate (0.5% w/v) for P. putida.

or pJRDEE32d13 were as low as that in the wild-type strain. Similar phenomenon was also observed in the transconjugants of P. putida GPp104. When phaPCAc genes were cointroduced into GPp104, a large amount of PHA synthase (Figure 4B) and detectable levels of the synthase activity were detected in the cells. Both strains of FA440/pJRDEE32d3 and GPp104/pJRDEE32d3, in which phaJAc was deleted from the pha locus, exhibited much lower enoyl-CoA hydratase activities than those harboring pJRDEE32. Nevertheless, these strains accumulated copolyesters with a high 3HHx fraction (4652 mol %) from alkanoates. This result indicated that the overexpression of phaJAc was not a reason for the remarked increase of 3HHx fractions in the copolyesters produced by the transconjugants harboring pJRDEE32. We further attempted the individual expression of phaPAc or phaCAc gene in A. caViae FA440 in order to identify which gene plays an important role for the compositional change. However, unfortunately, a high level expression of phaPAc or phaCAc using the native phaAc promoter or E. coli trc promoter could not be achieved in both cases for unknown reasons. We therefore constructed a new plasmid harboring phaPAc together with the mutant gene of phaCAc (referred to phaC′Ac) encoding PHA synthase with a point mutation of Cys319 to Ala. Cys319 in PHA synthase from R. eutropha has been identified as a catalytic center for the transfer of (R)-3-hydroxyacyl molecules to a propagating polyester chain,6 and the corresponding Cys residue is also conserved in the synthase from A. caViae at the same position.26 The resulting plasmid pJRDEE32d3A could not complement PHA-negative phenotype of P. putida GPp104 (Table 2) and R. eutropha PHB-4 (data not shown), indicating complete inactivation of the synthase by C319A mutation. A. caViae FA440 harboring pJRDEE32d3A highly expressed PhaPC′Ac

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Table 2. Accumulation of PHA and Enzyme Activities in Recombinant Strains of A. caviae FA440 and P. putida GPp104a strain

A. caviae FA440

P. putida GPp104

plasmids (relevant markers)

PHA content (wt %)

3HHx fraction (mol %)

PHA synthaseb (U/g)

enoyl-CoA hydratasec (U/mg)

none pJRDEE32 (phaPCJAc) pJRDEE32d1 (phaCJAc) pJRDEE32d3 (phaPCAc) pJRDEE32d13 (phaCAc) pJRDG13 (phaPJAc) pJRDEE32d3A (phaPC′Ac)d pJRDEE32 (phaPCJAc) pJRDEE32d1 (phaCJAc) pJRDEE32d3 (phaPCAc) pJRDEE32d13 (phaCAc) pJRDEE32d3A (phaPC′Ac)d

35 51 32 48 32 29 50 43 2 31 2 0

16 58 16 46 15 21 32 30 13 51 17 n.d.e

52 568 21 519 20 23 292 26 >10 125 >10 n.d.

43 360 61 66 49 47 n.d. 110 43 14 16 n.d.

a Cells were cultivated for 36 h at 30 °C in an MS medium containing sodium dodecanote (0.25% w/v) for A. caviae strains or sodium octanoate (0.5% w/v) for P. putida strains. b Activity toward (R)-3HB-CoA. c Activity toward crotonyl-CoA. d phaC′Ac, a gene encoding PHA synthase with C319A mutation. e n.d., not determined.

Table 3. Substrate Specificity of PHA Synthase Bound on Native PHA Inclusions

PHA inclusions from (genotype of strain)a FA440 (phaPCJAc) FA440/pJRDEE32 (phaP+C+J+Ac)

3HHx composition in PHA inclusion fract (mol %) 13 60

PHA synthase on granules sp. activity (U/g of dry ratio substrateb inclusion) C6/C4 (R)-3HB-CoA (R)-3HHx-CoA (R)-3HB-CoA (R)-3HHx-CoA

10 1.7 106 21

pJRDEE32 (0.17 and 0.20, respectively). The overexpression of phaPCJAc in A. caViae did not affect the substrate specificity of the synthase bound on native PHA inclusions. It is noteworthy that FA440/pJRDEE32 accumulated copolyester with a high 3HHx fraction (60 mol %) despite the much higher polymerization activity toward (R)-3HB-CoA than toward (R)-3HHx-CoA.

0.17 0.20

a Key: none, chromosome encoded; +, chromosome and vector encoded. b Generated in situ from the corresponding trans-2-enoyl-CoA by (R)-specific enoyl-CoA hydratase.

proteins when grown on dodecanoate, as well as FA440/ pJRDEE32d3 (Figure 4, lane7). Interestingly, FA440/ pJRDEE32d3A exhibited more than a 6-fold higher PHA synthase activity compared with the wild-type strain FA440, although the phaC′Ac on pJRDEE32d3A encodes an inactive PHA synthase. In addition, this recombinant strain accumulated P(3HB-co-3HHx) copolyester from dodecanoate with higher 3HHx fraction (32 mol %) than the wild-type strain (Table 2). Substrate Specificity of PHA Synthase Bound on PHA Inclusions. The native P(3HB-co-3HHx) inclusions composed of 13 and 60 mol % of 3HHx fraction were isolated from FA440 and the transconjugant harboring pJRDEE32, respectively, and PHA synthase activities of the inclusion fractions were assayed toward (R)-3HB-CoA and (R)3HHx-CoA. These (R)-isomers were generated in situ from the corresponding trans-2-enoyl-CoA by the function of (R)specific enoyl-CoA hydratase purified from recombinant E. coli harboring phaJAc.27 The results are shown in Table 3. PHA synthase activities toward (R)-3HB-CoA and (R)3HHx-CoA could be detected in these inclusion fractions, indicating that the PHA synthase of A. caViae is actually capable of accepting (R)-3-hydroxyacyl-CoA of C4 and C6 as substrates. The inclusion fraction from FA440/pJRDEE32, where a larger amount of PHA synthase bound (Figure 1, lane 6), exhibited 10-fold higher polymerization activity than that from FA440. However, the ratio of activities toward the substrates with different chain length (C6/C4) were similar in both the inclusion fractions from FA440 and FA440/

Discussion We have previously reported that PHA biosynthesis genes of A. caViae, a P(3HB-co-3HHx)-producer, is organized as a gene cluster consisting of ORF1 with unknown function, and genes of PHA synthase and (R)-specific enoyl-CoA hydratase.26,27 The ORF1 gene was here revealed to encode a 13 kDa granule-associated protein (GA13), so ORF1 was designated as phaPAc (Figure 2). PhaP are nonenzymatic granule-associated proteins identified in various bacteria,9-19 and have been proposed to be amphiphilic molecules that separate the hydrophobic core of the PHA inclusions from the hydrophilic cytoplasm. It has been known that the expression levels of phaP affect the content and size of PHA inclusions.13 Also, in our case, A. caViae FA440 highly expressing phaPAc synthesized numerous number of small inclusion bodies (Figure 3). PhaPAc shared 36.8% identity to that from Acinetobacter sp., but showed low homology toward those from R. eutropha and R. ruber (10.0% and 13.0%, respectively). The hydrophobic or amphiphilic regions for interaction with PHA inclusions, which has been proposed in PhaP from R. ruber, were not conserved in the primary structure of PhaPAc. These PhaP proteins seems to have no phylogenetic relation despite the similar function. High level activity of PHA synthase in recombinant A. caViae and P. putida could be achieved only when phaPAc was coexpressed with phaCAc. In addition, the introduction of pJRDEE32d3A carrying phaPC′Ac into A. caViae resulted in the increase of PHA synthase activity, as shown in Table 2, even though C319A mutant of PHA synthase from phaC′Ac on the plasmid was completely inactivated. This result suggested the additional function of PhaPAc for the activation of PHA synthase, as well as association with PHA inclusions. Although detail for the activation is still unclear, there are two possibilities for this phenomenon. One possibility is that

Compositional Change of PHA in Recombinant Bacteria

PhaPAc might be a positive transcriptional regulator for phaAc genes. Recently, Prieto et al. has proposed that the translated product of phaF, having AAKP repeat units like histone H1, is not only a class of granule-associated proteins but also has a regulatory function repressing the transcription of phaC1 and itself in P. oleoVorans.18 Contrary to the pseudomonad strain, excess PhaPAc might enhance the transcription of chromosomal phaCAc gene in A. caViae cells. Other possibility is that PhaPAc might activate PHA synthase by protein-protein interaction at the surface of PHA inclusions. Further studies will be needed to elucidate the function of PhaPAc. It is noteworthy that the protein components on PHA granules isolated from A. caViae are very simple (only PHA synthase and GA13) in comparison with those from other bacteria.11,13,15,17 This fact would be an advantage for investigation of the functions of PhaPAc. When alkanoates were used as a carbon source, A. caViae has been proved to generate (R)-3HB-CoA and (R)-3HHxCoA monomers from the corresponding trans-2-enoyl-CoA intermediates of β-oxidation by the function of (R)-specific enoyl-CoA hydratase (PhaJAc).27 Then PhaCAc subsequently accepts the C4-C6 monomers to elongate a polyester chain. A. caViae FA440/pJRDEE32, a strain highly expressing phaPCJAc, accumulated P(3HB-co-3HHx) with a much higher 3HHx fraction than the wild-type strain, as described above. A similar tendency was also seen in the case of P. putida GPp104 used as a heterologous host (Table 2). Despite the quite different PHA compositions, the substrate specificities of PHA synthase in the inclusion fraction, that is the ratio of synthase activity for (R)-3HB-CoA to (R)-3HHxCoA, were very similar between FA440 and FA440/ PJRDEE32 (Table 3). These results not only ruled out a possibility that the overexpressed PhaPAc changed the specificity of PhaCAc on the inclusion surface (increase of affinity toward a longer 3HHx monomer, or decrease of affinity toward a shorter 3HB monomer) but also suggested that the substrate specificity of PHA synthase was not a major factor for determination of the PHA composition. In addition, FA440/pJRDEE32d3 also accumulated copolyesters with high 3HHx fraction, indicating that overexpression of phaJAc was not related to the significant increase of the 3HHx fraction. This was probably because the hydration step catalyzed by PhaJAc was not a rate-limiting step in the pathway by A. caViae. The major difference between the transconjugants accumulating PHA with high 3HHx fraction and the wild-type strain was the level of PHA synthase activity. As shown in Table 2, the 3HHx fractions in the copolyesters were correlated with the PHA synthase activities in various recombinant strains of A. caViae. These results suggested that the level of PHA synthase activity may be an important factor for copolyester biosynthesis via β-oxidation. The enhanced PHA synthase activity might intercept and pull the longer C6 intermediates of β-oxidation into copolyester biosynthesis, prior to further degradation into shorter C4 intermediates. Such modification of metabolic flow might result in the drastic increase of the 3HHx unit and the corresponding decrease of the 3HB unit in the copolyesters. Further investigation, such as in vitro polymerization using purified PHA synthase, will be needed to explain this phenomenon.

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The recombinant bacteria, FA440/pJRDEE32 and GPp104/ pJRDEE32d3, were able to produce P(3HB-co-3HHx) copolyesters with high 3HHx fractions (60-70 mol %), which could not be synthesized by wild-type strain of A. caViae. These results obtained here indicate the usefulness of recombination techniques for production of PHAs with altered molar composition. Acknowledgment. This work was supported by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST). References and Notes (1) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (2) Doi, Y. Microbial polyesters; VCH Publishers: New York, 1990. (3) Steinbu¨chel, A. In Biotechnology; Rehm, H. J., Reed, G., Eds.; VCH Publishers: Weinheim, Germany, 1996; Vol. 6, p 403. (4) Madison, L. L.; Huisman, G. W. Microbiol. Mol. Biol. ReV. 1999, 63, 21. (5) Rehm, B. H. A.; Steinbu¨chel, A. Int. J. Biol. Macromol. 1999, 25, 3. (6) Gerngross, T. U.; Snell, K. D.; Peoples, O. P.; Sinskey, A. J.; Csuhai, E.; Masamune, S.; Stubbe, J. Biochemistry 1994, 33, 9311. (7) Zhang, S.; Takagi, Y.; Lenz, R, W.; Goodwin, S. Biomacromolecules 2000, 1, 244. (8) Muh, U.; Sinskey, A. J.; Kirby, D. P.; Lane, W. S.; Stubbe, J. Biochemistry 1999, 38, 826. (9) Steinbu¨chel, A.; Aerts, K.; Babel, W.; Fo¨llner, C.; Liebergesell, M.; Madkour, M. H.; Mayer, F.; Pieper-Fu¨rst, U.; Pries, A.; Valentin, H. E.; Wieczorek, R. Can. J. Microbiol. 1995, 41, 94. (10) Wieczorek, R.; Steinbu¨chel, A.; Schmidt. B. FEMS Microbiol. Lett. 1996, 135, 23. (11) Pieper-Fu¨rst, U.; Madkour, M. H.; Mayer, F.; Steinbu¨chel, A. J. Bacteriol. 1994, 176, 4328. (12) Liebergesell, M.; Steinbu¨chel, A. Biotechnol. Lett. 1996, 18, 719. (13) Wieczorek, R.; Pries, A.; Steinbu¨chel, A.; Mayer, F. J. Bacteriol. 1995, 177, 2425. (14) Hanley, Z. S.; Pappin, D. J. C.; Rahman, D.; White, A. J.; Elborough, K. M.; Slabas, A. R. FEBS Lett. 1999, 447, 99. (15) Schembri, M. A.; Woods, A. A.; Bayly, R. C.; Davies, J. K. FEMS Microbiol. Lett. 1995, 133, 277. (16) Maehara, A.; Ueda, S.; Nakano, H.; Yamane, T. J. Bacteriol. 1999, 181, 2914. (17) McCool, G. J.; Cannon, M. C. J. Bacteriol. 1999, 181, 585. (18) Prieto, M. A.; Bu¨hler, B.; Jung, K.; Witholt, B.; Kessler, B. J. Bacteriol. 1999, 181, 858. (19) Valentin, H. E.; Stuart, E. S.; Fuller, R. C.; Lenz, R. W.; Dennis, D. J. Biotechnol. 1998, 64, 145. (20) Kichise, T.; Fukui, T.; Yoshida, Y.; Doi, Y. Int. J. Biol. Macromol. 1999, 25, 69. (21) Huijberts, G. N. M.; Eggink, G.; de Waard, P.; Huisman, G. W.; Witholt, B. Appl. EnViron. Microbiol. 1992, 58, 536. (22) Huisman, G. W.; Wonink, E.; de Koning, G.; Preusting, H.; Witholt, B. Appl. Microbiol. Biotechnol. 1992, 38, 1. (23) Kraak, N. K.; Smits, T. H. M.; Kessler, B.; Witholt, B. J. Bacteriol. 1997, 179, 4985. (24) Doi, Y.; Kitamura, S.; Abe. H. Macromolecules 1995, 28, 4822. (25) Shimamura, E.; Kasuya, K.; Kobayashi, G.; Shiotani, T.; Shima, Y.; Doi, Y. Macromolecules 1994, 27, 878. (26) Fukui, T.; Doi, Y. J. Bacteriol. 1997, 179, 4821. (27) Fukui, T.; Shiomi, N.; Doi, Y. J. Bacteriol. 1998, 180, 667. (28) Huisman, G. W.; Wonink, E. W.; Meima, R.; Kazemier, B.; Terpstra, P.; Witholt, B. J. Biol. Chem. 1991, 266, 2191. (29) Schlegel, H. G.; Lafferty, R.; Krauss, I. Arch. Mikrobiol. 1970, 71, 283. (30) Simon, R.; Priefer, U.; Pu¨hler, A. Bio/Technology 1983, 1, 784. (31) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (32) Deng, W. P.; Nickoloff, J. A. Anal. Biochem. 1992, 200, 81. (33) Kato, M.; Bao, H. J.; Kang, C. K.; Fukui, T.; Doi, Y. Appl. Microbiol. Biotechnol. 1996, 45, 363. (34) Fukui, T.; Yokomizo, S.; Kobayashi, G.; Doi, Y. FEMS Microbiol. Lett. 1999, 170, 69.

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