Compositional regulation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by replacement of granule-associated protein in Ralstonia eutropha
© Kawashima et al. 2015
Received: 3 September 2015
Accepted: 4 November 2015
Published: 23 November 2015
Phasin (PhaP), a kind of polyhydroxyalkanoate (PHA) granule-associated proteins, has a role in controlling the properties of PHA granules surface, and is thought to have influence on PHA biosynthesis in PHA-producing bacteria. This study focused on the phaP1 Re locus in Ralstonia eutropha as a site of chromosomal modification for production of flexible poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] from soybean oil.
Considering the high expression level of phaP1 Re , phaJ Ac [encoding (R)-specific enoyl-CoA hydratase from Aeromonas caviae] was inserted into the downstream of phaP1 Re on chromosome 1 of R. eutropha strain NSDG harboring phaC NSDG (encoding PHA synthase with broad substrate specificity). The constructed strain efficiently accumulated P(3HB-co-3HHx) on soybean oil with higher 3HHx composition when compared to the previous strain having phaJ Ac within pha operon. Insertion of the second phaC NSDG along with phaJ Ac at the phaP1 Re locus led to incorporation of much larger 3HHx fraction into PHA chains, although the molecular weight was markedly reduced. The R. eutropha strains were further engineered by replacing phaP1 Re with phaP Ac (encoding phasin from A. caviae) on the chromosome. Interestingly, the phasin replacement increased 3HHx composition in the soybean oil-based PHA with keeping high cellular contents, nevertheless no modification was conducted in the metabolic pathways. Kinetic and Western blot analyses of PHA synthase using cellular insoluble fractions strongly suggested that the phasin replacement not only enhanced activity of PHA synthase from A. caviae but also increased affinity especially to longer (R)-3HHx-CoA. It was supposed that the increased affinity of PHA synthase to (R)-3HHx-CoA was responsible for the higher 3HHx composition in the copolyester.
The downstream of phaP1 Re was a useful site for integration of genes to be overexpressed during PHA accumulation in R. eutropha. The results also clarified that polymerization properties of PHA synthase was affected by the kind of phasin co-existed on the surface of PHA granules, leading to altered composition of the resulting P(3HB-co-3HHx). The phasin replacement is a novel engineering strategy for regulation of composition of PHA copolyesters.
KeywordsPolyhydroxyalkanoates Ralstonia eutropha PHA synthase Phasin
Polyhydroxyalkanoates (PHAs) are biopolyesters produced by a number of microorganisms as intracellular carbon and energy storage materials. PHAs have attracted industrial attentions as one of possible solutions for recent environmental problems caused by general petroleum-based plastics, because PHAs are eco-friendly polymeric materials that can be produced from renewable biomass resources, and show biodegradable and biocompatible properties [1, 2].
In PHA-accumulating bacterial cells, PHA chains are gathered and make granules in cytoplasm, and the PHA granules are covered with various proteins called PHA granule-associated proteins, PGAPs [15, 16]. Phasins (PhaPs) are amphiphilic small size proteins and known to be major PGAPs widely distributed in various PHA producers [17, 18]. The functions of PhaPs are thought to not only control the properties of PHA granules surface, but also have influence on PHA biosynthesis. PhaP1 Re is the most abundant phasin in R. eutropha H16 . The expression level is very high owing to the strong phaP1 promoter [19, 20], by which transcription is regulated by PHA granule-binding transcriptional factor PhaR Re [15, 21]. PhaP1 Re occupied approximately 5 % of the total proteins in the crude extract of the cells cultivated on fructose , and was estimated to cover 27–54 % of surface of the PHA granules . It was demonstrated that PhaP1 Re participated in control of the size and number of intracellular PHA granules and consequent amount of PHA, as the phaP1 Re -deficient strain of R. eutropha stored approximately half amount of P(3HB) as many smaller granules in comparison with the wild strain [17, 23]. Interestingly, PhaP1 Re did not bind to PhaC1 Re directly in two-hybrid assay , but formed a high-molecular weight complex along with PhaC1 Re and soluble P(3HB) oligomer in R. eutropha . The PhaC1 Re -PhaP1 Re -PHB complex showed no lag phase in PhaC activity assay, which suggested that PhaC Re had an active form in the complex.
Aeromonas caviae also has phasin (PhaP Ac ) of which gene is organized as phaP-C-J Ac operon on the chromosome . PhaP Ac belongs to a different class of phasin from PhaP1 Re , as they share no significant homology. When phaPCJ Ac genes were highly expressed in A. caviae, a numerous number of small PHA granules were accumulated within the cells . Moreover, a recombinant strain of A. caviae overexpressing phaPC Ac synthesized P(3HB-co-3HHx) with much higher 3HHx composition (46–52 mol %) than the strain overexpressing phaC Ac alone (16–21 mol %) . Although the detail for this phenomenon has not been elucidated, the result suggested some effects of PhaP Ac on catalytic properties of PhaC Ac . Recent in vitro analyses demonstrated that DNA/PHA-binding protein PhaM Re activated polymerization activity of PhaC1 Re , and formed a high-molecular-weight complex with PhaC1 Re . Another enzyme assay indicated that PhaP1 Re and PhaP Ac inhibited PhaC1 Re activity, whereas they enhanced the activity of PhaC Ac up to 2.4 to 3-fold .
In this study, we focused on the phaP1 Re locus as a site for chromosomal modification in R. eutropha for production of P(3HB-co-3HHx). The effects of insertion of PHA biosynthesis genes at the downstream of phaP1 Re , as well as those of replacement of phaP on biosynthesis of PHA copolyester from soybean oil were investigated.
Effects of insertion of phaJ Ac /phaC NSDG at downstream of phaP1 Re on PHA biosynthesis
PHA biosynthesis by R. eutropha recombinant strains from soybean oil
Dry cell mass (g/L)
PHA content (wt%)
3HHx composition (mol %)
(phaP1 Re strains)
6.9 ± 0.02
86 ± 4.3
1.6 ± 0.03
6.7 ± 0.21
91 ± 0.3
1.8 ± 0.10
6.7 ± 0.05
89 ± 1.5
6.5 ± 0.09
5.7 ± 0.18
84 ± 1.3
10.5 ± 0.26
6.8 ± 0.02
88 ± 0.51
4.6 ± 0.07
(phaP Ac strains)
7.1 ± 0.18
89 ± 1.1
2.0 ± 0.05
6.4 ± 0.11
88 ± 1.5
2.1 ± 0.51
7.0 ± 0.40
87 ± 1.1
8.8 ± 0.24
6.3 ± 0.07
79 ± 3.3
17.2 ± 0.18
7.2 ± 0.30
90 ± 1.2
6.4 ± 0.10
When phaC NSDG-phaJ Ac genes were inserted at the site, the resulting strain NSDG-P1ReCJ accumulated P(3HB-co-3HHx) with 3HHx fraction of 10.5 mol % (Table 1). This was 6.6-fold larger than that in the copolymer synthesized by the strain NSDG, although the PHA production was decreased from 5.9 to 4.8 g/l.
Effects of replacement of phaP1 Re by phaP Ac on PHA biosynthesis
phaP1 Re on chromosome 1 was replaced by phaP Ac from A. caviae in the recombinant strains of R. eutropha expressing phaC NSDG (Fig. 2), in order to investigate the change of PHA biosynthesis profiles by phasin derived from the same source as PHA synthase. Interestingly, this genetic modification tended to increase 3HHx composition without serious reduction of PHA production from soybean oil in all the strains examined, as shown in Table 1. The compositional change was noticeable in the strains expressing phaJ Ac and the additional phaC NSDG at the downstream of phaP, as the 3HHx fraction in P(3HB-co-3HHx) significantly increased to 17.2 mol % in NSDG-PAcCJ compared to 10.5 mol % in NSDG-P1ReCJ. The 3HHx compositions of the copolymers synthesized by NSDG-PAcJ and MF02-PAc (6.4–8.8 mol %) were also higher than those in the corresponding strains having native PhaP1 Re (4.6–6.5 mol %). The effect of the phasin replacement was not remarkable in the strains not expressing phaJ Ac , such as NSDG and NSDG-P1 Re C.
Molecular weights of PHAs accumulated in R. eutropha recombinant strains
Molecular weights of PHA synthesized by R. eutropha recombinant strains from soybean oil
M n × 105
M w × 105
7.5 ± 3.1
24.4 ± 5.23
3.56 ± 0.80
13.5 ± 2.3
42.3 ± 5.93
3.16 ± 0.26
10.0 ± 2.1
33.8 ± 6.05
3.52 ± 0.90
13.8 ± 1.2
48.1 ± 2.16
3.52 ± 0.25
1.2 ± 0.17
2.8 ± 0.25
2.28 ± 0.21
1.5 ± 0.36
3.6 ± 1.39
2.35 ± 0.32
Kinetic parameters of PHA synthase on the surface of PHA granules
Kinetic parameters of PhaCNSDG in insoluble fractions of R. eturopha recombinant strains toward (R)-3HB-CoA (C4) and (R)-3HHx-CoA (C6)
Substrate chain length
6.4 × 10−4
(phaP1 Re -phaC NSDG-phaJ Ac )
6.6 × 10−5
6.1 × 10−3
(phaP Ac -phaC NSDG-phaJ Ac )
1.8 × 10−3
Confirmation of phasin replacement and identification of proteins in PHA granule fractions
The proteins in PHA granule fractions of the strains NSDG-P1ReCJ and NSDG-PAcCJ, prepared by density-gradient ultracentrifugation, were separated by SDS-PAGE (Fig. 3b), and the major eight protein bands were subjected to LC–MS/MS analysis for identification. The details of the results are shown in Additional file 1: Table S1. The 24-kDa protein (no. 5) in NSDG-P1ReCJ was determined to be PhaP1 Re . In the strain NSDG-PAcCJ, this band was disappeared and a smaller protein (no. 8) in large abundance, not present in NSDG-P1ReCJ, was identified to be PhaC Ac . This result clearly indicated actual replacement of major phasin on PHA granules to PhaP Ac in NSDG-PAcCJ. The protein band no. 3 was outer membrane protein (porin), and the bands of no. 1, 2, 4, 6, and 7 contained multiple kinds of proteins including various membrane proteins, ribosomal proteins, and hypothetical proteins. Several proteins involved in important cellular functions were also detected, such as elongation factor Tu and cell division protein FtsA in the no. 1 band and RNA polymerase subunit α in the no. 2 band. These proteins were considered to be the result of contamination of other cell fractions, but possibility of the presence on the surface as PGAPS could not be excluded, as observed and discussed previously [16, 31]. Although R. eutropha possesses many phasins [3, 24, 29, 30], only PhaP5 Re could be detected in the band no. 7 besides PhaP1 Re in NSDG-P1ReCJ.
The gene of the major phasin in R. eutropha H16, phaP1 Re , is one of the most highly expressed genes at the PHA accumulation phase [19, 20]. Barnard et al. have achieved T7 promoter-driven high level production of recombinant proteins by using the phaP1 Re locus as the integration site for T7 RNA polymerase . We here applied the downstream of phaP1 Re as an insertion site for PHA biosynthesis-related gene(s) in R. eutropha, because the induction profile and expression level of phaP1 Re higher than phaCAB1 Re were expected to be advantage for PHA biosynthesis. When phaC NSDG was solely inserted at the site in the strain NSDG, no change in P(3HB-co-3HHx) composition was observed, probably due to poor provision of (R)-3HHx-CoA through the intact channeling pathway from β-oxidation to PHA biosynthesis. Indeed, enhancement of the channeling pathway by expression of phaJ Ac significantly increased 3HHx composition without impairment of PHA production, as observed previously [10, 12]. The results of the cultivation suggested that the expression level of the gene located in the phaP1 Re locus was higher than within pha operon, given the higher 3HHx composition in the resulting P(3HB-co-3HHx) and higher enoyl-CoA hydratase activity in NSDG-P1ReJ (phaP1 Re -phaJ Ac ) than those in the strain MF02 (phaC NSDG-phaJ Ac -phaAB1 Re ). The insertion of the second copy of phaC NSDG at the downstream of phaP1 Re led to drastic decrease of the molecular weight of PHA by less than one-tenth. Previous studies [33–35] reported a negative correlation of intracellular PhaC activity or phaC expression level with P(3HB) molecular weight, and one of possible reasons for this phenomenon may be due to large number of catalytic molecules (PhaCNSDG) relative to substrate molecules [(R)-3HA-CoAs]. The present study was consistent with the previous observations, and the similar effects of plasmid-borne multi copies of phaC on P(3HB-co-3HHx) biosynthesis has been also seen in recombinant R. eutropha grown on plant oils .
The expression of phaJ was very effective for increasing 3HHx composition in P(3HB-co-3HHx) synthesized from fatty acids and plant oils by recombinant R. eutropha strains [10–12]. However, we previously observed saturation of the provision of 3HHx unit by this strategy . Another strategy for increasing 3HHx composition was suppression of 3HB unit formation by disruption of phaA encoding thiolase, although this accompanied reduction of PHA production . The advantage of the present phasin replacement strategy was further increase of 3HHx composition even in the phaJ-expressing strains without impairment of PHA biosynthesis. The R. eutropha strains applying this novel strategy can efficienty produce P(3HB-co-3HHx) composed of 9–17 mol % 3HHx fractions, which was suitable for practical applications with adequate flexibility .
This study demonstrated that the downstream of phaP1 Re on chromosome 1 of R. eutropha was a useful site for integration of PHA biosynthesis genes to achieve the overexpression during the PHA accumulation phase. In addition, we found that replacement of the major phasin PhaP1 Re in recombinant R. eutropha by PhaP Ac from A. caviae resulted in increase of 3HHx composition of P(3HB-co-3HHx) without serious decrease of the productivity on soybean oil.
A general engineering strategy for alteration of PHA copolymer composition has been modification of metabolic flux of monomer-supplying pathways. The phasin replacement is a novel strategy not directly modifying metabolic pathways, thus there is no need to consider unexpected negative effects on global metabolisms. It was also shown that catalytic properties of PHA synthase were affected by phasin co-existed on the surface of PHA granules, and significance of the effects appeared to be depended on the kind of phasin. These results were interesting in understanding the role of phasin in PHA biosynthesis. In terms of the application, the use of adequate pair of PHA synthase and phasin would allow us to obtain PHA copolymers with desired composition with high productivity.
Bacterial strains and plasmids
The bacterial strains and plasmids used in this study are listed in Additional file 1: Table S2. R. eutropha strains were cultivated at 30 °C in a nutrient-rich (NR) medium containing 10 g of meat extract, 10 g of polypeptone, 2 g of yeast extract in 1 L of tap water. E. coli strains were cultivated at 37 °C in a Lysogeny broth (LB) medium. Kanamycin (100 mg/L) or ampicillin (50 mg/L) was added to the medium when necessary.
Construction of plasmids and strains
DNA manipulations were carried out according to standard procedures, and the sequences of oligonucleotide primers used for PCR are shown in Additional file 1: Table S3. KOD-Plus-ver.2 or KOD-Plus-Neo DNA polymerase and Ligation High ver.2 (Toyobo, Otsu, Japan) were used for PCR amplification and ligation, respectively.
The upstream and downstream regions flanked to phaP1 Re were individually amplified by PCR from R. eutropha H16 genomic DNA as a template with primer sets of phaPReout-Fw/phaPRe-Inv1 and phaPReout-Rv/phaPRe-Inv2, respectively. The latter fragment was 5′-phosphorylated, ligated with the former, and the upstream–downstream fragment was subjected to the second PCR using phaPReout-Fw/phaPReout-Rv primers. The amplified upstream–downstream fusion was inserted into pK18mobsacB at the SmaI site, leading to construction of pK18mobsacB-ΔP1.
The vectors for replacement of phaP1 Re by phaP Ac , phaP Ac -phaC NSDG, phaP Ac -phaC NSDG-phaJ Ac , or phaPJ Ac were constructed as follows. The coding region of phaC NSDG was amplified from pTA2-NSDG  by PCR using a primer set of pEE32R-CAcup-inv/pEE32R-CAcdown-inv. A linear fragment not containing phaC Ac in pEE32 harboring phaPCJ Ac  was prepared by inverse PCR with phaPRe-Inv1/phaPRe-Inv2 primers, and then ligated with the phaC NSDG fragment to obtain pEE32-NSDG harboring phaP Ac -C NSDG-J Ac . The fragments of phaP Ac , phaP Ac -C NSDG, and phaP Ac -C NSDG-J Ac were amplified with pEE32-NSDG as a template and primer sets of phaPAc-Fw/phaPAc-Rv, phaPAc-Fw/phaCNSDG-Rv, and phaPAc-Fw/phaJAc-Rv, respectively. The amplified fragments were 5′-phosphorylated and then individually inserted between upstream and downstream regions of phaP1 Re by ligation with an inverse PCR product obtained with pK18mobsacB-ΔP1 as a template and phaPRe-Inv1/phaPRe-Inv2 primers. The resulting plasmids were designated pK18mobsacB-PAc, pK18mobsacB-PAcC, and pK18mobsacB-PAcCJ, respectively. pK18mobsacB-PAcJ was constructed by inverse PCR for elimination of the phaC NSDG region from pK18mobsacB-PAcCJ with a primer set phaPAcdown-Inv/phaJAc-Fw, followed by self-ligation after 5′-phosphorylation.
pK18mobsacB-P1ReC, pK18mobsacB-P1ReJ and pK18mobsacB-P1ReCJ, used for insertion of the genes from A. caviae at downstream of phaP1 Re , were constructed from pK18mobsacB-PAcC, pK18mobsacB-PAcJ and pK18mobsacB-PAcCJ, respectively. Linear fragments not having the phaP Ac region were prepared from these plasmids by inverse PCR using phaPRe-Inv1/phaPAc-down-Inv primers. The coding region of phaP1 Re was amplified with R. eutropha H16 genome DNA and a primer set of phaPRe-Fw/phaPRe-Rv, and the phaP1 Re fragment was ligated with the corresponding inverse PCR product with the same direction as the phaP1 promoter.
All the pK18mobsacB-based plasmids were transferred from E. coli S17-1 by conjugation to R. eutropha strains NSDG or MF02, and the recombinant strains formed by pop-in-pop-out recombination were selected, according to the procedures reported previously .
PHA production by recombinant R. eutropha strains
PHA production by R. eutropha strains was carried out on a reciprocal shaker (115 strokes/min) at 30 °C in a 500 ml flask with a 100 ml of a nitrogen-limited mineral salts (MB) medium and 0.1 ml of trace-element solution . Soybean oil was directly added to the medium at 1.0 % (v/v) as a sole carbon source. After the cultivation for 72 h, the cells were harvested, washed with cold 70 % ethanol, washed again with deionized water, and then lyophilized. The cellular PHA content and monomer composition were determined by gas chromatography (GC) after methanolysis of the dried cells in the preference of 15 % sulfuric acid . Extraction and purification of the accumulated PHA from the dried cells, and determination of molecular weight by gel permutation chromatography (GPC) were performed as described previously .
Crotonyl-CoA was synthesized by condensation of crotonic anhydride (Tokyo Chemical Industry, Tokyo, Japan) with lithium salt of CoA-SH . Chemical synthesis of (R)-3HB-CoA and trans-2-hexenoyl-CoA by a mixed-anhydride method, and enzymatic conversion of trans-2-hexenoyl-CoA to (R)-3HHx-CoA were performed as described previously , except for the use of recombinant PhaJ4a Re  as (R)-specific enoyl-CoA hydratase.
Ralstonia eutropha strains were cultivated for 48 h in an MB medium at 30 °C with 1.0 % (v/v) soybean oil as a carbon source. The grown cells were harvested, washed by Tris–HCl buffer (pH 7.5), and then disrupted by high pressure homogenization as described previously . The disrupted cells were centrifuged (18,800g, 10 min, 4 °C) to separate the soluble fraction and insoluble fraction containing native PHA granules. PHA synthase activity of the insoluble fraction was assayed with the reaction mixture composed 0.05–0.5 mM (R)-3HB-CoA or (R)-3HHx-CoA, 1 mM 5,5′-dithiobis (2-nitrobenzoic acid) [DTNB] in 50 mM phosphate buffer (pH 7.2), and 10 µl of the insoluble fraction. The increase in absorbance at 412 nm corresponding to release of free CoA-SH was measured spectrophotometrically at 30 °C (ε412 = 14.5 × 103). Enoyl-CoA hydratase activity, including both (S)- and (R)-specific activities, in the soluble fraction was determined in the reaction mixture composed of 250 µM crotonyl-CoA or trans-2-hexenoyl-CoA in 50 mM Tris–HCl (pH 8.0) and 5 µl of the soluble fraction. The hydration of the enoyl-CoA substrates were monitored as decrease in absorbance at 263 nm at 30 °C (ε263 = 6.7 × 103 M−1 cm−1). (R)-specific enoyl-CoA hydratase activity in the soluble fraction was determined by the hydration of the enoyl-CoA substrates coupled with polymerization of the resulting (R)-3-hydroxyalyl-CoAs of C4 and C6 by using the insoluble fraction of R. eutropha NSDG-PAcC as a source of PHA synthase. The reaction mixture was composed of 0.20 mM trans-2-enoyl-CoA, 1 mM DTNB in 50 mM phosphate buffer (pH 8.0), 5 µl of the insoluble fraction of the strain NSDG-PAcC, and the increase of absorbance at 412 nm was monitored at 30 °C.
The aliquot of the insoluble fraction was lyophilized to determine the dry weight, and used to calculate specific activities. The proteins contained in 12.5 µg of the insoluble fraction were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 15 % acrylamide gel, according to the standard procedure. Western blot analysis for PhaCNSDG was performed using a specific antiserum against PhaC Ac . A goat anti-rabbit IgG (Fc fragment specific)-alkaline phosphatase conjugate (Calbiochem, CA, USA) was used as the secondary antibody, and 1-Step NBT/BCIP plus suppressor (Thermo Fisher Scientific, MA, USA) was used to detect the signals according to the manufacture’s instruction. Protein concentration of the soluble fraction was determined by the method of Bradford with bovine serum albumin as the standard.
Transmission electron microscope
The cells of the R. eutropha strains cultivated in an MB medium with 1 % (v/v) soybean oil at 30 °C for 48 h were harvested, washed, and resuspended in 0.1 M phosphate buffer (pH 7.2) containing 0.1 M sucrose. The cells were fixed with 2.5 % (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) containing 0.1 M sucrose, and subsequently with 1 % (w/v) osmium tetroxide in the buffer. The fixed cells were dehydrated in a graded ethanol and embedded with epoxy resin Quetol 651 (Nissin EM, Tokyo, Japan). The ultrathin sections (80 nm) of the embedded cells were prepared with a Leica UC7 ultramicrotome. The sections on a copper grid were stained with EM stainer (Nissin EM) followed by Reynolds’s lead citrate at room temperature. The specimens were observed on a JEOL 1400Plus transmission electron microscope with an accelerating voltage of 100 kV.
Isolation of native PHA granule fractions and identification of proteins
The insoluble fractions of R. eutropha strains were loaded on the top of a discontinuous glycerol gradient of 2 mL of 88 % glycerol and 8 mL of 44 % glycerol in 100 mM Tris–HCl (pH 7.5). After the ultracentrifugation (210,000g, 40 min, 4 °C), the PHA granules at the interphase of 44–88 % glycerol were collected, following by washing with 100 mM Tris–HCl (pH 7.5). The granules resuspended in 1 mL of the buffer were subsequently loaded on a discontinuous sucrose gradient prepared with 1 mL of 1.8 M sucrose, 2.9 mL of 1.6, 1.4, and 1.2 M sucrose, and 1 mL of 1.0 M sucrose in the buffer, and then subjected to ultracentrifugation (210,000g, 2 h, 4 °C). The band of the PHA granules were observed at the interphase of 1.2–1.4 M sucrose for P(3HB-co-3HHx) composed of 10.5 mol % 3HHx (produced by the strain NSDG-P1ReCJ), and that of 1.0–1.2 M sucrose for P(3HB-co-3HHx) composed of 17.2 mol % 3HHx (produced by the strain NSDG-PAcCJ). The bands of PHA granules were recovered, washed, and resuspended in 100 mM Tris–HCl (pH 7.5).
The PHA granule suspension containing approximately 700 μg of dried inclusion was separated by SDS-PAGE with 4–15 % gradient gel (Bio-Rad, Hercules, CA, USA). The tryptic peptides were prepared from the SDS-PAGE gel, and subjected to mass spectrometric analysis using an LC–MS system equipped with Acquity UPLC apparatus (Waters, Milford, MA, USA) and Synapt High Definition Mass Spectrometer (Waters). LC was performed with a reversed-phase C18 column (75 µm × 150 mm, 1.7 µm particle size) with the mobile phase of acetonitrile gradient in 1 % formic acid at the flow rate of 0.5 μl/min. The mass spectrometry was done in the positive ion mode. The MS/MS spectra were analyzed by Protein Lynx Global Server (Waters).
YK carried out the experiments and data analyses, and wrote the manuscript. IO and SN participated in the design and coordination of the study. TF designed the experiments and prepared the manuscript. All authors read and approved the final manuscript.
We thank Assoc. Prof. T. Tsuge and Dr. A. Hiroe (Department of Innovative and engineered Materials, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, Japan) for GPC analysis. We also thank Assoc. Prof. N. Hayashi (Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan) for identification of PGAPs by LC–MS/MS, Ms. K. Ikeda and Bio-technical center of Tokyo Institute of Technology for TEM analysis, and Ms. M. Segawa for her kind assistance of synthesis of the substrates used in enzyme assay. This work was supported by JSPS KAKENHI Grant Numbers 25630373 and 26011384.
YK, IO, and TF are listed as inventors on a PCT application covering the phasin replacement for PHA biosynthesis described in this publication.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Taguchi S, Iwata T, Abe H, Doi Y. Poly(hydroxyalkanoate)s. In: Matyjaszewski K, Möller M, editors. Polymer science: a comprehensive reference. Amsterdam: Elsevier;2012. p. 157–82.Google Scholar
- Keshavarz T, Roy I. Polyhydroxyalkanoates: bioplastics with a green agenda. Curr Opin Microbiol. 2010;13:321–6.View ArticleGoogle Scholar
- Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E, Strittmatter A, Voss I, Gottschalk G, Steinbüchel A, Friedrich B, Bowien B. Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16. Nat Biotechnol. 2006;24:1257–62.View ArticleGoogle Scholar
- Reinecke F, Steinbüchel A. Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. J Mol Microbiol Biotechnol. 2008;16:91–108.View ArticleGoogle Scholar
- Doi Y, Kitamura S, Abe H. Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules. 1995;28:4822–8.View ArticleGoogle Scholar
- Fukui T, Doi Y. Cloning and analysis of the poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) biosynthesis genes of Aeromonas caviae. J Bacteriol. 1997;179:4821–30.Google Scholar
- Fukui T, Shiomi N, Doi Y. Expression and characterization of (R)-specific enoyl coenzyme a hydratase involved in polyhydroxyalkanoate biosynthesis by Aeromonas caviae. J Bacteriol. 1998;180:667–73.Google Scholar
- Tsuge T, Watanabe S, Shimada D, Abe H, Doi Y, Taguchi S. Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis. FEMS Microbiol Lett. 2007;277:217–22.View ArticleGoogle Scholar
- Mifune J, Nakamura S, Fukui T. Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can J Chem. 2008;86:621–7.View ArticleGoogle Scholar
- Mifune J, Nakamura S, Fukui T. Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polym Degrad Stab. 2010;95:1305–12.View ArticleGoogle Scholar
- Budde CF, Riedel SL, Willis LB, Rha C, Sinskey AJ. Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains. Appl Environ Microbiol. 2011;77:2847–54.View ArticleGoogle Scholar
- Kawashima Y, Cheng W, Mifune J, Orita I, Nakamura S, Fukui T. Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha. Appl Environ Microbiol. 2012;78:493–502.View ArticleGoogle Scholar
- Insomphun C, Mifune J, Orita I, Numata K, Nakamura S, Fukui T. Modification of β-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil. J Biosci Bioeng. 2014;117:184–90.View ArticleGoogle Scholar
- Insomphun C, Xie H, Mifune J, Kawashima Y, Orita I, Nakamura S, Fukui T. Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha. Metab Eng. 2015;27:38–45.View ArticleGoogle Scholar
- Pötter M, Steinbüchel A. Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules. 2005;6:552–60.View ArticleGoogle Scholar
- Jendrossek D, Pfeiffer D. New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3-hydroxybutyrate). Environ Microbiol. 2014;16:2357–73.View ArticleGoogle Scholar
- Wieczorek R, Pries A, Steinbüchel A, Mayer F. Analysis of a 24-kilodalton protein associated with the polyhydroxyalkanoic acid granules in Alcaligenes eutrophus. J Bacteriol. 1995;177:2425–35.Google Scholar
- Wieczorek R, Steinbbchel A, Schmidt B. Occurrence of polyhydroxyalkanoic acid granule-associated proteins related to the Alcaligenes eutrophus H16 GA24 protein in other bacteria. FEMS Microbiol Lett. 1996;135:23–30.View ArticleGoogle Scholar
- Brigham CJ, Budde CF, Holder JW, Zeng Q, Mahan AE, Rha C, Sinskey AJ. Elucidation of β-oxidation pathways in Ralstonia eutropha H16 by examination of global gene expression. J Bacteriol. 2010;192:5454–64.View ArticleGoogle Scholar
- Shimizu R, Chou K, Orita I, Suzuki Y, Nakamura S, Fukui T. Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses. BMC Microbiol. 2013;13:169–83.View ArticleGoogle Scholar
- Pötter M, Madkour MH, Mayer F, Steinbüchel A. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 2002;148:2413–26.View ArticleGoogle Scholar
- Tian J, He A, Lawrence AG, Liu P, Watson N, Sinskey AJ, Stubbe J. Analysis of transient polyhydroxybutyrate production in Wautersia eutropha H16 by quantitative western analysis and transmission electron microscopy. J Bacteriol. 2005;187:3825–32.View ArticleGoogle Scholar
- York GM, Stubbe J, Sinskey AJ. New insight into the role of the PhaP phasin of Ralstonia eutropha in promoting synthesis of polyhydroxybutyrate. J Bacteriol. 2001;183:2394–7.View ArticleGoogle Scholar
- Pfeiffer D, Jendrossek D. Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16. Microbiology. 2011;157:2795–807.View ArticleGoogle Scholar
- Cho M, Brigham CJ, Sinskey AJ, Stubbe J. Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo. Biochemistry. 2012;51:2276–88.View ArticleGoogle Scholar
- Fukui T, Kichise T, Iwata T, Doi Y. Characterization of 13 kDa granule-associated protein in Aeromonas caviae and biosynthesis of polyhydroxyalkanoates with altered molar composition by recombinant bacteria. Biomacromolecules. 2001;2:148–53.View ArticleGoogle Scholar
- Pfeiffer D, Jendrossek D. PhaM is the physiological activator of poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha. Appl Environ Microbiol. 2014;80:555–63.View ArticleGoogle Scholar
- Ushimaru K, Motoda Y, Numata K, Tsuge T. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase. Appl Environ Microbiol. 2014;80:2867–73.View ArticleGoogle Scholar
- Pötter M, Müller H, Reinecke F, Wieczorek R, Fricke F, Bowien B, Friedrich B, Steinbüchel A. The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology. 2004;150:2301–11.View ArticleGoogle Scholar
- Pfeiffer D, Jendrossek D. Localization of poly(3-hydroxybutyrate) (PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16. J Bacteriol. 2012;194:5909–21.View ArticleGoogle Scholar
- Sznajder A, Pfeiffer D, Jendrossek D. Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16. Appl Environ Microbiol. 2015;81:1847–58.View ArticleGoogle Scholar
- Barnard GC, Henderson GE, Srinivasan S, Gerngross TU. High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification. Protein Expr Purif. 2004;38:264–71.View ArticleGoogle Scholar
- Kahar P, Tsuge T, Taguchi K, Doi Y. High yield production of polyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombinant strain. Polym Degrad Stab. 2004;83:79–86.View ArticleGoogle Scholar
- Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T. Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl Environ Microbiol. 2012;78:3177–84.View ArticleGoogle Scholar
- Sim SJ, Snell KD, Kim BW, Rha CK, Sinskey AJ. Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli. Biotechnol Lett. 2001;23:2057–61.View ArticleGoogle Scholar
- Kato M, Bao HJ, Kang C-K, Fukui T, Doi Y. Production of a novel copolyester of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids by Pseudomonas sp. 61-3 from sugars. Appl Microbiol Biotechnol. 1996;45:363–70.View ArticleGoogle Scholar
- Fukui T, Mukoyama M, Orita I, Nakamura S. Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates. Appl Microbiol Biotechnol. 2014;98:7559–68.View ArticleGoogle Scholar
- Simon EJ, Shemin D. The preparation of S-succinyl coenzyme A. J Am Chem Soc. 1953;75:2520.View ArticleGoogle Scholar
- Fukui T, Chou K, Harada K, Orita I, Nakayama Y, Bamba T, Nakamura S, Fukusaki E. Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16. Metabolomics. 2013;10:190–202.Google Scholar