Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstraße 3, D-48149 Münster, Germany1
Institut für Organische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 40, D-48149 Münster, Germany2
Author for correspondence: Alexander Steinbüchel. Tel: +49 251 8339821. Fax: +49 251 8338388. e-mail: steinbu{at}uni-muenster.de
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ABSTRACT |
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Keywords: polyhydroxyalkanoate, polythioester, Ralstonia eutropha, 3-mercaptopropionic acid, 3,3'-thiodipropionic acid
Abbreviations: 3HB, 3-hydroxybutyrate; 3HP, 3-hydroxypropionate; 3MP, 3-mercaptopropionic acid (as constituent of the polymer); GPC, gel permeation chromatography; PHA, polyhydroxyalkanoate; PHB, poly(3-hydroxybutyrate); TDP, 3,3-thiodipropionic acid
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INTRODUCTION |
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Although a large number of different PHAs have been detected, neither the biosynthesis of PHAs with sulfur in the backbone nor a biological polythioester has been described so far. Only recently, the incorporation of a thiophenoxy group at the end of the side chain into PHAs was reported (Takagi et al., 1999 ). In this study, we report for the first time the bacterial production of a copolyester consisting of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP). The incorporation of the hitherto undescribed constituent, 3MP, is catalysed by an enzymic reaction resulting in a thioester bond. Therefore, poly(3HB-co-3MP) can be designated as a representative of an eighth class of biological polymers: polythioesters (Table 1
).
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METHODS |
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Fed-batch cultivation of R. eutropha H16 on a 26 l scale was done in a stirred (at 200400 r.p.m.) and aerated (1520 l min-1) 30 l stainless-steel fermenter (Biostat UD30, B. Braun, Biotech International). Fermentations were carried out in MSM, and the pH was adjusted to 7·0.
Polymer isolation from lyophilized cells.
Poly(3HB) and poly(3HB-co-3MP) were extracted from lyophilized cells with chloroform, filtered, precipitated in 10 vols ethanol, and dried under a constant air stream. In order to obtain highly purified polymer, the precipitation procedure was repeated at least threefold.
GC/MS analysis.
The polyester content was determined by methanolysis of 57 mg lyophilized cells in the presence of sulfuric acid, and the resulting methyl esters were characterized by GC (Brandl et al., 1988 ). For molecular analysis of the methyl esters, a coupled GC/MS was performed using an HP 6890 gas chromatograph equipped with a model 5973 mass selective detector (Hewlett Packard). The mass spectra obtained were compared with the NIST 98 Mass Spectral Library with Windows Search Program version 1.6, National Institute of Standards and Technology (US Department of Commerce).
Elemental sulfur analysis.
Sulfur analysis was performed by the Mikroanalytisches Labor Beller (Göttingen, Germany) according to the method of Grote & Krekeler (Deutsches Institut für Normung DIN 51768).
Molecular mass analysis.
The molecular masses of purified polyesters were estimated by gel-permeation chromatography (GPC) relative to polystyrene standards (990, 810, 500, 280, 198, 120 and 85 kDa). Analysis was performed on four Styragel columns (HR 3, HR 4, HR 5, HR 6 with pore sizes of 103, 104, 105 and 106 ; 1
=0·1 nm) connected in line in a Waters GPC apparatus. Samples were eluted with chloroform at a flow rate of 1·0 ml min-1 and at 35 °C, and the eluted compounds were monitored by a Waters 410 differential refractometer. Polydispersity and the number average (MN) and weight average (MW) molecular masses were calculated by using the Millenium Chromatography Manager GPC software (Waters).
IR spectroscopic analysis.
The IR spectra were taken with a Fourier transform spectrometer IFS 28 (Bruker). The samples were dissolved in CHCl3 and deposited as a film on a sodium chloride disk. Alternatively, a liquid cell with sodium chloride windows (path length 0·5 mm) was used with a chloroform solution of the polymer (2 mg sample ml-1).
IR spectrum of poly(3HB-co-3MP) (cm-1); film on NaCl disk.
2983 m (CH,CH2,CH3); 2933 m (CH,CH2,CH3); 1737 s (ester C=O valence); 1688 (thioester C=O valence); 1380 m; 1302 m; 1260 m (CH2S); 1185 s (ester CO); 1134 m; 1101 m; 1057 s; 978 m; 760 w; 700 w.
NMR spectroscopic analysis.
All NMR experiments were performed with a Varian Unity Plus 600 spectrometer (1H, 599·14 MHz; 13C, 150·66 MHz). The 1H and 13C assignments were confirmed through gCOSY (gradient 1H,1H-COSY), 1D TOCSY (1H total correlation spectroscopy with selective excitation), gHSQC (1H, 13C gradient heteronuclear single quantum coherence) and gHMBC (1H, 13C gradient heteronuclear multiple bond correlation) spectra. The measurements were carried out at 298 K with a sample of 10 mg of the isolated polymer dissolved in 1 ml CDCl3. While the three investigated polymer samples (see Table 2) showed very close values for the sets of chemical shifts, there were some variations regarding the amount of incorporated 3MP. Therefore, in the following the NMR spectroscopic results of one representative polymer originating from the 50 h fed-batch fermentation of R. eutropha (Table 2
) are listed in the order of determined sequence types.
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3MP3MP: (1H) 3·13 (m, 3J=7 Hz, 2H, 3-H), 2·84 (m, 3J=7 Hz, 2H, 2-H);
(13C) 196·7 (Cq, C-1, thioester), 43·3 (CH2, C-2), 24·0 (CH2, C-3); gHMBC: 2·84/196·7, 3·13/196·7.
3HB3MP: (1H) 5·27 (m, 1H, 3-H), 2·86 (m, 1H, 2-H), 2·70 (m, 1H, 2-H'), 1·27 (d, 3H, 4-H);
(13C) 195·2 (Cq, C-1, thioester), 67·8 (CH, C-3), 49·4 (CH2, C-2), 19·8 (CH3, C-4); gHMBC: 2·86/195·2, 2·70/195·2, 5·27/195·2.
3HB3MP: (1H) 3·10 (m, 3-H); gHMBC: 3·10/195·2.
3MP3HB: (1H) 3·10 (m, 2H, 3-H), 2·58 (m, 2H, 2-H);
(13C) 170·5 (Cq, C-1, ester), 34·4 (CH2, C-2), 24·0 (CH2, C-3); gHMBC: 2·58/170·5, 3·10/170·5.
3HP3HB: (1H) 4·30 (m, 2H, 3-H), 2·59 (m, 2H, 2-H);
(13C) 60·0 (CH2, C-3), 33·9 (CH2, C-2); gHMBC: 4·30/169·8.
3HP3MP: (1H) 4·35 (m, 2H, 3-H), 2·88 (m, 2H, 2-H);
(13C) 195·8 (Cq, C-1, thioester), 60·0 (CH2, C-3), 43·7 (CH2, C-2); gHMBC: 2·88/195·8, 4·35/195·8, 4·35/171·3.
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RESULTS AND DISCUSSION |
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Growth experiments employing mineral salts medium containing 3-mercaptopropionic acid or TDP plus a carbon source that is readily utilized, such as gluconic acid, revealed that 3-mercaptopropionic acid at concentrations higher than 0·1% (w/v) in the medium impaired the growth of R. eutropha and other bacteria (data not shown). In contrast to 3-mercaptopropionic acid, TDP did not exert any toxic effects on the growth of R. eutropha up to concentrations of 1·5% (w/v).
Chemical analysis of poly(3HB-co-3MP)
GC analyses of cells from R. eutropha cultivated with 3-mercaptopropionic acid and TDP in addition to fructose or gluconic acid under conditions promoting PHA accumulation showed peaks with a retention time of RT=8·88 min, in addition to the 3HB methyl ester at a RT of 9·55 min (Fig. 2). The polymer was isolated from the cells and highly purified; all subsequent analyses were performed with the purified polyester. Three batches of purified polymer obtained from three different fermentations (Table 2
) were analysed. The peaks were analysed by MS, and the 3MP-methyl ester was identified by the isotope pattern (Fig. 2
). Comparison with the NIST database confirmed the identification of the 3MP methyl ester as an acid methanolysis product of poly(3HB-co-3MP).
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Molecular mass analyses of poly(3HB-co-3MP)
The molecular masses of the isolated polymers were determined by GPC relative to polystyrene standards. The weight average molecular mass (MW) of poly(3HB-co-3MP) varied from 0·49 to 1·12x106 g mol-1. The polydispersity indices (MW/MN) ranged from 1·1 to 7·0, with unimodal distribution (Table 2). Compared with the homopolyester poly(3HB) synthesized by R. eutropha from gluconic acid under conditions permissive for PHA accumulation, the molecular masses of poly(3HB-co-3MP) correlated with those of poly(3HB) reported previously (Rehm & Steinbüchel, 1999
).
IR spectroscopic analysis of poly(3HB-co-3MP)
The IR spectrum reflects both monomeric units. All absorptions due to the PHB moiety appeared in the spectrum, and in addition a strong absorption band at 1688 cm-1 was detected, as is expected for the C=O valence vibration of a thioester bond (Colthup et al., 1964 ) (Fig. 3
). The intensity of this band was proportional to the sulfur content, which was determined by elemental analysis, as is shown in the inset in Fig. 3
.
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NMR spectroscopic analysis of poly(3HB-co-3MP)
Fig. 4 shows the 600 MHz 1H-NMR spectrum of a typical poly(3HB-co-3MP) sample. The fraction of 3MP present in the polymer was determined by integration of the 3-H signals of 3HB and 3MP resonating at
5·23 and 3·23, respectively. The calculated incorporation rate of 34% is in good agreement with the 34·9% determined by elemental sulfur analysis for this polymer sample (Table 2
). In addition, traces of 3-hydroxypropionic acid (3HP) resonating at
4·30 were detected (see below).
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Interestingly, 3MP-CoA is obviously used as substrate by the PHA synthase and, surprisingly, the PHA synthase is obviously able to catalyse the formation of both oxoester and thioester bonds. The PHB-negative mutant R. eutropha PHB-4 was not capable of synthesizing poly(3HB-co-3MP), confirming the involvement of PHA synthase. The broad substrate range of PHA synthases, as indicated by the different carbon chain length and by the occurrence of various substituents at the alkyl moiety, is well known (Steinbüchel & Valentin, 1995 ). The proposed catalytic mechanism of the PHA synthase in R. eutropha involves two thiol groups which derive from two cysteine residues of the enzyme subunits (PhaC) forming a homodimer (Müh et al., 1999
; Rehm & Steinbüchel, 1999
) (Fig. 8
). These thiol groups covalently bind the growing polyester chain, and the constituent that will be incorporated during the next turn of the cycle. A nucleophilic attack of the free electron pair of the hydroxy group of the latter at the carbonyl carbon atom of the nascent polymer is suggested (Griebel et al., 1968
; Wodzinska et al., 1996
; Müh et al., 1999
). The thiol group of 3MP can obviously also provide a free electron pair for this nucleophilic attack, and 3MP is incorporated, resulting in the formation of a thioester (Fig. 8
).
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Conclusions and perspectives
Poly(3HB-co-3MP) is the first representative of a new class of biopolymers which are designated as polythioesters (Table 1). In addition, it is the first biopolymer described that contains sulfur in the polymer backbone. The only other sulfur-containing biopolymers known are proteins, some complex polysaccharides, and very recently described PHAs containing thiophenoxy groups (Takagi et al., 1999
); however, they contain sulfur in the side chains. This study reveals a promising basis for further basic research and new technical applications. The thermoplastic and/or elastomeric features of PHAs allow various applications and uses in industry, e.g. in the packaging industry, medicine, pharmacy, agriculture or the food industry considering the clear advantages of biodegradability and origin from renewable resources (Hocking & Marchessault, 1994
). Only recently, polythioesters containing 3MP or other constituents, and polyesters containing TDP, were chemically synthesized (Podkoscielny & Podgorski, 1996
; Bandiera et al., 1997
; Choi et al., 1998
; Kameyama et al., 1999
). Some interesting properties of these polymers were revealed, and they were suitable for preparing polymer electrolytes (Bandiera et al., 1997
). The chemical and physical properties of poly(3HB-co-3MP) are now under investigation in order to reveal in particular the influence of the sulfur atoms on the properties of the polymer and possible modifications of the polymer such as cross-linking between the polymer chains. In addition, it may be expected that various other sulfur-containing constituents will also be incorporated into polymers by PHA synthases. Furthermore, it will be interesting to determine whether poly(3HB-co-3MP) is biodegradable and susceptible to hydrolytic attack by PHA depolymerases or lipases as are PHAs (Jaeger et al., 1995
; Jendrossek et al., 1996
).
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ACKNOWLEDGEMENTS |
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Received 29 June 2000;
revised 28 September 2000;
accepted 6 October 2000.