Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages

Tina Lütke-Eversloh1, Klaus Bergander2, Heinrich Luftmann2 and Alexander Steinbüchel1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
This is the first report on the biosynthesis of a hitherto unknown, sulfur-containing polyester and also the first report on a bacterial polymer containing sulfur in the backbone. The Gram-negative polyhydroxyalkanoate (PHA)-accumulating bacterium Ralstonia eutropha synthesized a copolymer of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP), when 3-mercaptopropionic acid or 3,3’-thiodipropionic acid was provided as carbon source in addition to fructose or gluconic acid under nitrogen-limited growth conditions. The peculiarity of this polymer was the occurrence of thioester linkages derived from the thiol groups of 3MP and the carboxyl groups of 3MP or 3HB, respectively, which occurred in addition to the common oxoester bonds of PHAs. Depending on the cultivation conditions and the feeding regime, poly(3HB-co-3MP) contributed up to 19% of the cellular dry weight, with a molar fraction of 3MP of up to 43%. The chemical structure of poly(3HB-co-3MP) was confirmed by GC/MS, IR spectroscopy, 1H- and 13C-NMR spectroscopy, and elemental sulfur analysis. The identification of this novel biopolymer reveals a new quality regarding the substrate range of PHA synthases and their capability for the synthesis of technically interesting polymers.

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


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Polymers are the most abundant molecules in living matter. Generally, seven classes of biopolymers are distinguished: polynucleotides, polyamides, polysaccharides, polyisoprenes, lignin, polyphosphate and polyhydroxyalkanoates (PHAs) (Müller & Seebach, 1993 ) (Table 1). Poly(3-hydroxybutyrate), PHB, belongs to the latter class as a widespread bacterial storage compound and was already observed in 1926 as hydrophobic inclusions in the cytoplasm of Bacillus megaterium (Lemoigne, 1926 ). Today many genera of bacteria are known to accumulate PHAs as energy and carbon source mostly under restricted growth conditions, e.g. oxygen or nitrogen limitation (Anderson & Dawes, 1990 ; Steinbüchel, 1991 ).


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Table 1. Eight classes of biopolymers: characteristics of their biosynthesis and occurrence

 
Bacteria synthesize PHAs from coenzyme A thioesters of the respective hydroxyalkanoic acid and are able to produce a wide range of different PHAs due to the rather unspecific PHA synthases that catalyse the polymerization reaction. In 1974, 3-hydroxyvaleric acid and 3-hydroxyhexanoic acid were identified as additional constituents of these bacterial polyesters (Wallen & Rohwedder, 1974 ). More than 130 different hydroxyalkanoic acids are now known as constituents of bacterial PHAs (for a review see Steinbüchel & Valentin, 1995 ). Only a few polyesters can be obtained from simple and abundantly available carbon sources, e.g. glucose. The large variety of PHAs comprises 3-, 4-, 5- and 6-hydroxyalkanoic acids of varying chain length, possibly containing additional methyl or other alkyl groups, double bonds, or different substituents at various positions of the hydroxyalkanoic acid, and is often based on the feeding of suitable precursor substrates, which exhibit chemical structures related to the PHA constituents (Steinbüchel & Valentin, 1995 ).

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).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and culture conditions.
Ralstonia eutropha H16 (DSM 428) and R. eutropha PHB-4 (DSM 541) were cultivated in nutrient broth (NB) growth medium or in mineral salts medium (MSM) (Schlegel et al., 1961 ) at 30 °C in Erlenmeyer flasks under aerobic conditions on a rotary shaker at 130 r.p.m. In order to promote PHA accumulation, the ammonia concentration was reduced to 0·05% (w/v). Carbon sources were added from filter-sterilized 20% (w/v) stock solutions at the concentrations indicated in the text.

Fed-batch cultivation of R. eutropha H16 on a 26 l scale was done in a stirred (at 200–400 r.p.m.) and aerated (15–20 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 5–7 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) {nu} (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 (CH2–S); 1185 s (ester C–O); 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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Table 2. Characterization of poly(3HB-co-3MP) obtained from different fermentations of R. eutropha

 
3HB3HB–: {delta}(1H) 5·23 (m, 1H, 3-H), 2·58 (dd, 2J=15·5 Hz, 3J2,3=7·3 Hz 1H, 2-H), 2·45 (dd, 2J=15·5 Hz, 3J2,3=6 Hz, 1H, 2-H'), 1·25 (d, 3J3,4=6·3 Hz, 3H, 4-H); {delta}(13C) 169·1 (Cq, C-1, ester), 67·6 (CH, C-3), 40·8 (CH2, C-2), 19·7 (CH3, C-4); gHMBC: 2·58/169·1, 2·45/169·1, 5·23/169·1.

3MP3MP–: {delta}(1H) 3·13 (m, 3J=7 Hz, 2H, 3-H), 2·84 (m, 3J=7 Hz, 2H, 2-H); {delta}(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–: {delta}(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); {delta}(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–: {delta}(1H) 3·10 (m, 3-H); gHMBC: 3·10/195·2.

3MP–3HB–: {delta}(1H) 3·10 (m, 2H, 3-H), 2·58 (m, 2H, 2-H); {delta}(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.

3HP–3HB–: {delta}(1H) 4·30 (m, 2H, 3-H), 2·59 (m, 2H, 2-H); {delta}(13C) 60·0 (CH2, C-3), 33·9 (CH2, C-2); gHMBC: 4·30/169·8.

3HP–3MP–: {delta}(1H) 4·35 (m, 2H, 3-H), 2·88 (m, 2H, 2-H); {delta}(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.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Growth and polymer accumulation by R. eutropha
Although R. eutropha strain H16 is not able to use 3-mercaptopropionic acid or 3,3'-thiodipropionic acid (TDP) as sole carbon sources for growth, a copolyester of 3HB and 3MP, poly(3HB-co-3MP) (Fig. 1), was synthesized when these substances were provided in addition to fructose or gluconic acid under nitrogen limitation. The PHB-negative mutant PHB-4 of strain H16 did not synthesize a polymer containing 3MP.



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Fig. 1. Structural formula of poly(3HB-co-3MP).

 
Table 2 summarizes the culture conditions, sulfur contents, polymer yields, molar fractions of 3MP, molecular masses and polydispersity indices of three different batches of the novel polymer. Depending on the culture conditions, the polymer content of the cells varied from 9·2 to 19·2% (w/w). Usually, R. eutropha accumulates poly(3HB) when the provided carbon source is directly metabolized to acetyl-CoA, and under certain conditions the polymer content can contribute up to 96% of the cellular dry weight (Pedrós-Alio et al., 1985 ). In this study, a decrease of total poly(3HB-co-3MP) content was observed simultaneously with an increase of the molar 3MP fraction when the utilizable carbon source was restricted. Thus, a molar fraction of 3MP of 42·5 mol% was obtained during batch cultivation of R. eutropha with nitrogen and gluconic acid limitation in the presence of 0·1% (w/v) 3-mercaptopropionic acid.

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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Fig. 2. GC/MS analysis of poly(3HB-co-3MP) from R. eutropha. The purified polymer was hydrolysed by acid, and the methyl esters obtained were separated by GC (a). For quantification, heptanoic acid, which occurred as a methyl ester (#1), was added to the samples as internal standard prior to esterification. Small amounts of S-methylmercaptopropionic acid methyl ester were observed, resulting in an additional peak with a retention time of RT=10·66 min (#2). The mass spectra of the 3MP moiety (b) and 3HB moiety (c) are also presented.

 
In order to determine the precise molar fraction of 3MP in the novel polymer, elemental sulfur analysis of the isolated and purified poly(3HB-co-3MP) was carried out. The total sulfur content varied from 9·96 to 15·67% (w/w); thus the calculated 3MP content varied from 26·9 to 42·5 mol% in the three polymer batches.

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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Fig. 3. IR spectrum of poly(3HB-co-3MP) from R. eutropha. The inset represents the correlation between the absorption of the thioester band and the 3MP content in the purified polymer.

 
The IR spectroscopic analysis gave further insights into the chemical structure without a previous hydrolysis of the polymer. PHAs with modified backbones have already been identified. These PHAs consisted of 2-methyl-3-hydroxybutyric acid (Satho et al., 1992 ; Füchtenbusch et al., 1996 ) or 3-hydroxypivalic acid (Füchtenbusch et al., 1998 ) and contained one or two methyl groups at the {alpha}-carbon atom. However, poly(3HB-co-3MP) is the first example of a polymer in which the linkage between the constituents is modified: the –3HB–3MP– and –3MP–3MP– are linked by thioester bonds that occur in addition to the oxoester linkages of –3MP–3HB– and –3HB–3HB–.

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 {delta} 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 {delta} 4·30 were detected (see below).



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Fig. 4. 600 MHz 1H-NMR spectrum of poly(3HB-co-3MP) from R. eutropha (CDCl3, 298 K).

 
In principle, there are four possibilities for combining the monomers. The homopolymer regions of 3HB and 3MP offer a relatively homogeneous environment. Accordingly, such sequences lead to sharp and intense signals in the 1H- and 13C-NMR spectra. The corresponding 1H and 13C signals in Figs 4 and 5, respectively, are labelled accordingly. The observed chemical shifts in the homopolymer regions of 3HB are in good agreement with the signal assignment of poly(3HB-co-3HP) (Shimamura et al., 1994 ), while the assignment of the homopolymer region of 3MP was done in comparison with the methyl ester of 3MP.



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Fig. 5. 150 MHz 13C-NMR spectrum of poly(3HB-co-3MP) from R. eutropha (CDCl3, 298 K).

 
In addition to these two types of linkages, the signals of –3HB–3MP– and –3MP–3HB–, containing thioester and ester bonds, respectively, can also be expected in the isolated polymer. These linkages can be traced by 1H, 13C long-range correlation spectra. Consequently, the gHMBC spectrum presented in Fig. 6 showed correlation signals between the protons of 3HB and the C-1 of the thioester, and of 3MP and C-1 of the ester. Compared with the signals of the homopolymer regions of 3HB and 3MP, these signals were of lower intensity. Because of signal overlap the exact molar relation between the homo- and the copolymer regions could not be determined. However, we were able to achieve definitive signal assignment through 1D TOCSY experiments by selective excitation at various resonance frequencies; the results are listed in Methods. Poly(3HB-co-3MP) is obviously neither a random copolymer nor a blockpolymer, but its structure can be described as ‘blocky’. In addition to these four main chain sequences, we found indications for the presence of small amounts of –3HP–3HB– and –3HP–3MP– as is shown in Figs 4 and 5. Although traces of 3HP were detected by NMR spectroscopic analyses, GC/MS analyses did not reveal any occurrence of 3HP or other constituents in the purified polymers.



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Fig. 6. 600 MHz 1H, 13C-gHMBC spectrum of poly(3HB-co-3MP) from R. eutropha (CDCl3, 298 K).

 
Considerations of the biosynthetic pathway of poly(3HB-co-3MP)
As a prerequisite for PHA biosynthesis, the provided carbon source must be transported into the cells, and is subsequently metabolized via central pathways (e. g. fatty acid ß-oxidation, citric acid cycle, fatty acid de novo synthesis) or special pathways to a hydroxyacyl coenzyme A thioester (Anderson & Dawes, 1990 ). In the simplest way, a hydroxyalkanoic acid can be directly activated to the corresponding coenzyme A thioester, which serves as substrate for PHA synthase, the key enzyme of PHA synthesis catalysing the polymerization reaction. Thus, uptake and activation of 3-mercaptopropionic acid to 3MP-CoA and subsequent incorporation into poly(3HB-co-3MP) is most likely to occur in R. eutropha if 3-mercaptopropionic acid is provided as carbon source (Fig. 7). The conversion of 3MP to 3MP-CoA has been shown for example in rat heart mitochondria, where it is catalysed by the medium-chain acyl-CoA synthetase (Cuebas et al., 1985 ). Inhibitory effects on enzymes of the ß-oxidation pathway in mitochondria caused by 3MP-CoA have been described (Sabbagh et al., 1985 ), possibly explaining the growth inhibition of R. eutropha and other bacteria due to higher concentrations of 3-mercaptopropionic acid in the media.



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Fig. 7. Putative pathway for the biosynthesis of poly(3HB-co-3MP) from 3,3’-thiodipropionic acid or 3-mercaptopropionic acid in R. eutropha.

 
Regarding the catabolism of TDP, we suppose an enzymic cleavage of TDP into 3MP and 3HP (Fig. 7), because 3MP amounted to up to 34·9 mol% of the constituents of the polymer obtained during fed-batch cultivation of R. eutropha with nitrogen and gluconic acid limitation in the presence of an excess of TDP. 3MP and 3HP are subsequently converted to the corresponding CoA thioesters. 3HP was identified previously as a constituent of PHAs synthesized by R. eutropha (Nakamura et al., 1991 ). However, the enzyme responsible for this cleavage has yet to be identified. Whether this reaction occurs by a reductive or a different mechanism remains to be elucidated.

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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Fig. 8. Putative catalytic cycle of the PHA synthase (PhaC) for the incorporation of 3MP into poly(3HB-co-3MP) in R. eutropha.

 
Although little or nothing is known about the degradation of the sulfur-containing substances TDP and 3-mercaptopropionic acid, a biological conversion into intermediates of central pathways is very likely to occur, because 3-mercaptopropionic acid is not a xenobiotic and occurs naturally. 3-Mercaptopropionic acid is a central intermediate in the catabolism of dimethylsulfoniopropionate (DMSP), an osmolyte in marine algae (van der Maarel & Hansen, 1997 ). So far, the isolation of bacteria growing with 3-mercaptopropionic acid as sole carbon source has failed, but experimental evidence for bacterial degradation of 3-mercaptopropionic acid in marine environments has been obtained (Kiene & Taylor, 1988 ). Therefore, the biosynthesis of a polymer structure, which may turn out to be the first identified member of a new class of naturally occurring biopolymers, is conceivable as well.

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 ).


   ACKNOWLEDGEMENTS
 
We thank Markus Pötter for performing the GPC analysis of the polymers.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 29 June 2000; revised 28 September 2000; accepted 6 October 2000.