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1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany
2 Integrierte Funktionelle Genomik, Westfälische Wilhelms-Universität Münster, Röntgenstraße 21, D-48149 Münster, Germany
3 Institut für Medizinische Physik und Biophysik, Universitätsklinikum, Westfälische Wilhelms-Universität Münster, Robert-Koch-Straße 31, D-48149 Münster, Germany
Correspondence
Alexander Steinbüchel
steinbu{at}uni-muenster.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISSCUSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISSCUSIONREFERENCES |
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. PTEs are synthesized by polyhydroxyalkanoate (PHA) synthases, which are the key enzymes for biosynthesis of the polyoxoesters poly(3-hydroxybutyrate) [poly(3HB)] or other PHAs (Taguchi & Doi, 2004) if bacteria are cultivated in the presence of organic thiochemicals yielding mercaptoacyl-coenzyme A thioesters by cell metabolism, thus indicating the low substrate specificity of these enzymes (Steinbüchel & Valentin, 1995). The Gram-negative bacterium Ralstonia eutropha accumulates copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP), 3-mercaptobutyrate (3MB) or 3-mercaptovalerate (3MV) when the cells are cultivated under conditions permissive for PHA biosynthesis in the presence of various thiochemicals (Lütke-Eversloh et al., 2001a, b; Lütke-Eversloh & Steinbüchel, 2003). A recombinant strain of Escherichia coli expressing the non-natural BPEC pathway, relying on the enzymes butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) from Clostridium acetobutylicum plus a two-component PHA synthase (PhaEC) from non-oxygenic photosynthetic bacteria like Allochromatium vinosum or Thiococcus pfennigii (Liu & Steinbüchel, 2000a, b), synthesized poly(3MP), poly(3MB) or poly(3MV) homopolymers when cultivated in the presence of the respective 3-mercaptoalkanoic acids (Lütke-Eversloh et al., 2002a). In addition, PTEs were also synthesized in vitro, employing the Candida antarctica lipase (Iwata et al., 2003; Kato et al., 2005).
PTEs exhibit interesting physical and biological properties. Thermal properties such as the melting point temperature and glass transition temperatures deviate significantly from those of the corresponding polyoxoester analogues (Lütke-Eversloh et al., 2002a, b; Kawada et al., 2003; Tanaka et al., 2004). For example, the melting point temperature (Tm) increases from 121 °C in poly(3HB) to 170 °C in poly(3MP). The PTE homopolymer poly(3MP) has recently become available in sufficient quantities by up-scaling the BPEC process to the 500 l scale and has been used for biodegradation studies (Thakor et al., 2005). Surprisingly, it turned out that poly(3MP) is non-biodegradable (Elbanna et al., 2004; Kim et al., 2005).
Transmission electron microscopic studies of thin sections of cells of the recombinant E. coli strain producing poly(3MP) show a large number of unusually small granules (Lütke-Eversloh et al., 2002a) in comparison to granules occurring, for example, in R. eutropha. This was surprising because the PTE biosynthesis pathway was expressed in the absence of a phasin protein. Phasins represent a class of small, amphiphilic structural proteins that bind to the surface of PHA granules and cover most of the surface (Wieczorek et al., 1995). These amphiphilic proteins constitute a boundary layer between the hydrophobic surface of the PHA granules owing to the amorphous hydrophobic polyester molecules and the mostly hydrophilic constituents of the cytoplasm. Thus they stabilize the ‘PHA in water dispersion’ in the cytoplasm, thereby yielding distinct, non-coalescing granules (Steinbüchel et al., 1995; Pötter & Steinbüchel, 2005). R. eutropha synthesizes four homologous phasin proteins (PhaP1, PhaP2, PhaP3 and PhaP4) with PhaP1 being the most abundant protein constituting about 3–5 % (w/w) of the total cell protein if the cells contain large amounts of PHAs (Wieczorek et al., 1995; Pötter et al., 2004). The number and size of PHA granules in cells depend very much on the presence of phasin proteins. In most cells of a phaP1 mutant of R. eutropha, only one single large and oval granule (length up to 2 µm) is seen, which occupies almost the entire cytoplasm, whereas wild-type cells accumulate several round granules of medium size (0.2–0.5 µm diam.). Furthermore, R. eutropha cells harbouring several copies of the phaP1 gene contain a much larger number of granules of small size. These findings provide evidence for the hypothesis of the ‘PHA in water emulsion’-stabilizing effect of phasins.
Since PTE and PHA molecules are not very different with regard to the hydrophobicity of the polymer molecules, the extraordinary small size of PTE granules in recombinant E. coli cells is not consistent with the hypothesis mentioned above. It indicates that other proteins could override the function of phasins in E. coli. Therefore, we investigated the proteins associated with PTE granules in recombinant E. coli strains expressing the BPEC pathway in the absence and presence of PhaP1 and of other R. eutropha phasins under conditions permissive for poly(3MP) or poly(3HB) biosynthesis and accumulation.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISSCUSIONREFERENCES |
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. E. coli was cultivated at 37 °C in 500 ml M9 mineral salts medium (Sambrook et al., 1989) supplemented with 0.01 % (w/v) yeast extract in 2 l Erlenmeyer flasks with baffles on a rotary shaker at 125 r.p.m. As carbon source, 1.0 % (w/v) glucose was added from a 20 % (w/v) filter-sterilized stock solution. After 12 h, 3-mercaptopropionic acid (99 %; Acros Organics) or sodium 3-hydroxybutyrate (Sigma–Aldrich Chemie) was added to the medium as the precursor substrate for poly(3MP) or poly(3HB) synthesis, respectively, to a concentration of 0.2 % (v/v or w/v, respectively). In addition, 1 mM IPTG was added to induce transcription of phaP1 on pCDFDuet-1. To maintain the plasmids, 75 µg ampicillin ml–1 was added to E. coli BL21 (DE3)/pBPP1 and 75 µg ampicillin ml–1 plus 50 µg streptomycin ml–1 to E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1.
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, employing Pfx DNA polymerase (Invitrogen) and an Omnigene HBTR3CM DNA thermal cycler (Hybaid). By using the Perfectprep Gel Cleanup Kit (Eppendorf), following the instructions described in the enclosed manual, the 579 bp fragment was purified from an agarose gel. The fragment was then digested with NcoI and HindIII, and was ligated into NcoI- and HindIII-digested pCDFDuet-1 vector DNA (Novagen), yielding pCDFDuet-1 : : phaP1.
Transfer of DNA.
Competent E. coli cells were prepared and transformed by the CaCl2 procedure as described by Hanahan (1983).
Analysis of PHAs and PTEs.
The polymer contents of the cells were determined upon methanolysis of 5–10 mg lyophilized cells in the presence of 85 % (v/v) methanol and 15 % (v/v) sulfuric acid. The resulting methyl esters of 3-HB and 3-MP were analysed by GC as described by Brandl et al. (1988) and Timm & Steinbüchel (1990).
Isolation of native PHA and PTE granules.
For the examination of granule-associated proteins, poly(3HB) and poly(3MP) granules were isolated by a modification of the method of Preusting et al. (1993) from E. coli cells which had been grown in M9 medium. Samples (50 ml) were withdrawn from the medium after 18, 24, 30 and 36 h incubation. The cells were harvested by centrifugation (20 min, 6000 g, 4 °C), washed in 100 mM Tris/HCl buffer (pH 8.0) and then suspended in 2 ml 100 mM Tris/HCl buffer (pH 8.0). After a threefold passage through a French press (100x106 Pa), the lysate was loaded on the top of a glycerol gradient. The gradient used for isolation of poly(3HB) granules was obtained from a discontinuous gradient prepared from 4 ml 90 % (v/v) plus 4 ml 60 % (v/v) glycerol in 100 mM Tris/HCl buffer (pH 8.0). The gradient for isolation of poly(3MP) granules was prepared from 3 ml 90 % (v/v), 3 ml 80 % (v/v) plus 3 ml 60 % (v/v) in 100 mM Tris/HCl buffer (pH 8.0). After centrifugation (1 h, 100 000 g, 4 °C), a granule layer of poly(3HB) was obtained at about 90 % (v/v) glycerol and a layer of poly(3MP) at about 80 % (v/v) glycerol. The granules were isolated from the gradients and then washed three times with Tris/HCl buffer (pH 8.0) by centrifugation (15 min, 16 100 g, 4 °C). The granules were stored at –20 °C for further analyses.
One-dimensional PAGE.
Protein samples were resuspended in gel loading buffer (0.6 %, w/v, SDS; 1.25 %, v/v, 
-mercaptoethanol; 0.25 mM EDTA; 10 %, v/v, glycerol; 0.001 %, w/v, bromophenol blue; 12.5 mM Tris/HCl, pH 6.8) and were separated in 12.5 % (w/v) SDS-polyacrylamide gels as described by Laemmli (1970). The proteins were stained with Coomassie brilliant blue R-250 (Weber & Osborn, 1969). Samples of crude extracts and of the native isolated granules were examined by this method.
Analysis of granule-associated proteins by MALDI-TOF MS.
Spots were excised from the PAGE gels, destained and washed using a slightly modified procedure to that described by Koltzscher et al. (2003). Proteins were tryptically digested in the gel, and peptides were extracted and C18-purified for MALDI-TOF MS. Peptide masses were measured using TofSpec-2E (Waters/Micromass). Database searches were performed with the Mascot engine in-house (Matrix Science) on Swiss-Prot, specifying E. coli proteins.
Electron microscopy studies.
To obtain transmission electron micrographs (TEM), cells were fixed with 2.5 % (v/v) glutaraldehyde in 0.1 M PBS (pH 7.3) immediately after they were withdrawn from the cultivation vessels. After three washing steps with 0.1 M PBS each for 20 min, the cells were post-fixed in 1 % (w/v) osmium tetroxide in 0.1 M PBS (pH 7.3) and washed once with the same PBS for 20 min. Then water was removed by a graded water/ethanol series (30, 50, 70, 90, 96 %, v/v, ethanol in water and absolute ethanol as final step), each step lasting for about 15 min. The following preparation steps were made according to the specific requirements of the microscopic method used. For thin sectioning, the samples were embedded in SPURR resin (without propylene oxide) (Spurr, 1969). Sections with a thickness of 70–80 nm were made with an Ultracut apparatus (Leica Mikroskopie und Systeme) using a diamond knife and were then positioned on a 200 mesh copper grid. Imaging was performed with an H-500 transmission electron microscope (Hitachi) in the bright-field mode at 75 kV acceleration voltage and at room temperature. Photographs were taken on Agfa-Gevaert 23 D 56 films.
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RESULTS AND DISSCUSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISSCUSIONREFERENCES |
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). To analyse the impact of PhaP1 on the accumulation of poly(3MP) in E. coli, we combined the genes encoding the non-natural BPEC pathway with phaP1. Moreover, the BPEC pathway was used to produce poly(3HB) in the presence and absence of PhaP1. For this, phaP1 was inserted into pCDFDuet-1, as described in Methods, yielding pCDFDuet-1 : : phaP1 (Fig. 1a). The genes for the BPEC pathway are encoded on the 11.96 kbp vector pBPP1 (Fig. 1b). Because of the compatibility of the two plasmids, they could both be transferred to and maintained in E. coli BL21 (DE3). In addition, E. coli BL21 (DE3) was transformed with pBPP1 only as a negative control.
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) and exhibit a PHA-leaky phenotype. Therefore, coexpression of PhaP1 in recombinant strains of E. coli capable of accumulating PTEs due to the expression of a PHA biosynthesis pathway might yield greater PTE content in the cells. The effect of phaP1 on poly(3HB) and poly(3MP) accumulation in E. coli cells expressing the non-natural BPEC pathway has not been investigated yet. To analyse the influence of PhaP1 from R. eutropha H16 on the accumulation of poly(3MP) and poly(3HB) in recombinant strains of E. coli expressing the non-natural BPEC pathway, E. coli BL21 (DE3)/pBPP1 and E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 were cultivated in M9 medium containing glucose. After 12 h cultivation, 3MP or 3HB was added and served as precursor substrates for poly(3MP) or poly(3HB) biosynthesis, respectively. After induction of expression of phaP1 by adding IPTG after 12 h, the cells were then further cultivated for 24 h. To obtain reliable results, all cultivations were done in duplicate. The growth behaviour of phasin-positive and -negative cultures was similar (Figs 2 and 3) and after about 18 h of cultivation, the cells reached stationary phase. Samples were withdrawn every 6 h and the poly(3HB) and poly(3MP) contents of the cells were analysed.
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; Liu & Steinbüchel, 2000b). In E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 cells, poly(3MP) contributed up to about 30 % (w/w) of the cell dry matter, whereas the poly(3MP) content of E. coli BL21 (DE3)/pBPP1 cells was only about 20 % (w/w) of the cell dry matter (Fig. 2
). The situation was similar when the cells were cultivated in the presence of 3HB. Whereas E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 cells accumulated poly(3HB) up to 27 % (w/w) of cell dry matter, this polymer amounted to only 17 % (w/w) of cell dry matter in E. coli BL21 (DE3)/pBPP1 cells (Fig. 3).
The results of these experiments clearly demonstrated that the major phasin PhaP1 of R. eutropha H16 exerts not only a positive effect on PHA accumulation in its own cells, but also if a PHA biosynthesis pathway is expressed in E. coli. The amounts of poly(3HB) synthesized by R. eutropha strains lacking intact PhaP by deletion of the gene or by Tn5 insertion are decreased by about 50 % compared to the wild-type (Wieczorek et al., 1995; York et al., 2002). It may be expected that similar positive effects also occur in other recombinant organisms expressing a PHA biosynthesis pathway. Moreover, this effect of PhaP1 is not restricted to biosynthesis and accumulation of polyoxoesters like poly(3HB), but also occurs with PTEs like poly(3MP) as clearly shown in this study. The positive effect of PhaP1 on polymer accumulation is significant, and the poly(3MP) and poly(3HB) contents of the cells could be increased by about 50 or 68 %, respectively. Both findings will be important for the optimization of strains suitable for biotechnological production of PHAs and PTEs. These results are in line with other studies, in which a positive effect of phasins on PHA biosynthesis and accumulation was also observed. Studies with the purified PhaEC of Allochromatium vinosum have demonstrated that the amount of in vitro-synthesized poly(3HB) can be significantly increased by the addition of purified PhaP1 (Jossek et al., 1998). Studies with the purified PhaC2 of Pseudomonas aeruginosa have shown that PhaP1 from R. eutropha increases the activity of the PHA synthase by about 50 % (Qi et al., 2000). It is possible that PhaP1 has the same influence on the activity of the PHA synthase PhaEC during poly(3MP) accumulation. Experiments with a recombinant strain of E. coli harbouring a plasmid encoding the phbCAB operon have shown that the accumulation of poly(3HB) can be increased from about 16 to 57 % of the cell dry matter when PhaP is also expressed (Seo et al., 2003). So far, unfortunately, a positive effect of phasins on PHA accumulation has not been demonstrated in transgenic plants. In transgenic Arabidopsis thaliana, coexpression of PhaP1 alongside PHB biosynthesis genes neither increases the polymer content nor alleviates the negative effect of expression of PHB biosynthesis on growth and development (Bohmert et al., 2002). To our knowledge, similar coexpression experiments have so far not been done in other transgenic plants.
Analysis of poly(3HB) and poly(3MP) granule-associated proteins
The protein patterns of crude extracts as well as those of isolated granules were analysed by PAGE in the same samples of E. coli BL21 (DE3)/pBPP1 and E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 cells cultivated in the presence of 3MP or 3HB that were analysed for their polymer contents (see above). The expression of PhaP1 could be demonstrated in cells of E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1. The electropherograms of crude extracts of cells of E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1, which were cultivated in the presence of 3MP, showed abundant amounts of a protein with an apparent molecular mass of 22 kDa representing PhaP1 (Fig. 4a). Similarly, crude extracts from cells of the same strain, which were cultivated in the presence of 3HB, showed a protein of identical size (Fig. 5a). This protein band was absent in crude extracts prepared from cells of E. coli BL21 (DE3)/pBPP1. Surprisingly, however, a protein exhibiting an apparent molecular mass of about 16 kDa occurred in the electropherograms of all crude extracts prepared from this E. coli strain which expressed the BPEC pathway but lacked phaP1, irrespective of whether the cells were cultivated in the presence of 3MP (Fig. 4a) or 3HB (Fig. 5a), respectively.
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and 5(b), respectively. This demonstrated that, in the absence of phasin PhaP1, this 16 kDa protein is expressed in E. coli and that it binds to the granules in the cytoplasm. MALDI-TOF analysis identified this 16 kDa protein unequivocally as the small heat-shock protein HspA (accession no. NP_756468) of E. coli CFT073. HspA is identical with the small heat-shock protein IbpA (accession no. P0C054) from E. coli. IbpA is expressed in E. coli in combination with another heat-shock protein, IbpB (accession no. P0C058), in situations where the cells are exposed to unfavourable conditions, for example during the overproduction of recombinant protein when it binds to inclusion bodies produced from recombinant proteins (Allen et al., 1992). The genes ibpA and ibpB constitute an operon, and the encoded proteins occur in complexes (Kuczy
ska-Wi
nik et al., 2002). IbpB is stimulated by IbpA to associate with a substrate (Matuszewska et al., 2005). While expressing recombinant proteins, E. coli synthesizes large amounts of IbpA and IbpB, and the proteins are bound to the inclusion bodies (Han et al., 2004). Interestingly, expression of these proteins to a detectable level and binding to the granules did not occur in E. coli expressing PhaP1. Obviously, HspA seems to act like a phasin and binds to the PHA and PTE granules yielding increased amounts of either polymer in the cells. The electropherograms indicate that, irrespective of whether E. coli BL21 (DE3)/pBPP1 cells were cultivated in the presence of 3MP or 3HB, they produce larger quantities of HspA than E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 cells produce PhaP1. Therefore, HspA is very abundant in the phasin-negative strains of E. coli. In recent studies, other larger heat-shock proteins, DnaK and GroEL/ES, have been detected in R. eutropha as granule-associated proteins when phaP1 was deleted (Pötter et al., 2004). These heat-shock proteins were also found in recombinant E. coli expressing the phbCAB operon (Han et al., 2001).
Electron microscopy studies
It is assumed that phasins bind to PHA granules not only to stabilize the dispersion of the hydrophobic polymer in the hydrophilic cytoplasm, thereby preventing individual granules from coalescing to a few granules or even a single large granule as observed in a phaP1 mutant in R. eutropha (Wieczorek et al., 1995), but probably also to prevent the unspecific binding of other proteins to the large surface of the granules, thereby avoiding misrouting of proteins. This will protect the cells from various forms of stress. Previously, it was shown, surprisingly, that recombinant E. coli cells which accumulated poly(3MP), deposit the accumulated polymer in a large number of very small granules, although the cells lack a phasin (Lütke-Eversloh et al., 2002a; Lütke-Eversloh & Steinbüchel, 2004). TEM images obtained in this study show that poly(3MP) granules isolated from E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 were even smaller (Fig. 6c, d) than those isolated from cells of the phasin-negative strain (Fig. 6a, b). The mean size of the poly(3MP) granules was only 55±12 nm in cells of E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 in comparison to the mean size of 105±12 nm in cells of E. coli BL21 (DE3)/pBPP1. Similarly, the mean size of poly(3HB) granules that accumulated in E. coli BL21 (DE3)/pBPP1+pCDFDuet-1 : : phaP1 was 56±10 nm (Fig. 7a, b), whereas the mean size of the poly(3HB) granules in the phasin-negative strain was 110±22 nm (Fig. 7c, d). This indicates that HspA could act like a phasin and, due to the large amounts of this heat-shock protein, coalescence of individual granules is widely, although not completely, prevented in the absence of PhaP1. Mutants with a defect in HspA will probably accumulate much larger granules or even just one single granule if, instead of HspA, the formation of a further protein which compensates for the loss of HspA is not induced.
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) and bovine serum albumin (Horowitz & Sanders, 1995). Analysis of the PHA granule proteome by two-dimensional gel electrophoresis and MALDI-TOF analysis revealed the presence of homologues to the heat-shock proteins DnaK and GroEL, and also the -ketothiolase Bkt in PHA granules isolated from cells of a phasin-negative mutant of R. eutropha (Pötter et al., 2004). However, the latter proteins were not bound to the granules of the wild-type, thus indicating that their binding is prevented by the phasin. In a study by Han et al. (2001), it was shown that the expression of the heat-shock proteins GroEL, GroES and DnaK was significantly up-regulated in a recombinant strain of E. coli expressing the Alcaligenes latus PHA biosynthesis genes; however, these proteins were detected only in crude extracts and no efforts were undertaken to analyse the PHA granule proteome in this E. coli strain. It has also been shown that the presence of these unspecifically bound proteins enhances the specific activity of the PHA synthase in vitro (Jossek et al., 1998). This and other studies indicate that the expression of PHA biosynthesis in E. coli causes stress to the cells and that significant changes occur in the proteome. E. coli synthesizes small heat-shock proteins to reduce stress by binding these proteins to recombinant expressed inclusion bodies. However, the influence of HspA is not equal to the influence of PhaP1 on poly(3HB) accumulation in R. eutropha H16, which can be observed in the different amounts of accumulated polymer. Because of the physical properties of PTEs and with regard to their industrial use, for example in medical or technical applications, these findings will be important for optimization of the biotechnological production of PTEs.
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ACKNOWLEDGEMENTS |
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Edited by: M. Hecker
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISSCUSIONREFERENCES |
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Lütke-Eversloh, T., Bergander, K., Luftmann, H. & Steinbüchel, A. (2001b). Biosynthesis of poly(3-hydroxybutyrate-co-3-mercaptobutyrate) as a sulfur analogue to poly(3-hydroxybutyrate) (PHB). Biomacromolecules 2, 1061–1065.[CrossRef][Medline]
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Received 28 June 2006;
revised 19 October 2006;
accepted 23 October 2006.
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