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: polyhydroxyalkanoates, Ralstonia eutropha, metabolic engineering, thia fatty acids, alkylthioalkanoate
Abbreviations: ATA, alkylthioalkanoic acid; BTV, butylthiovaleric acid; GC/MS, gas chromatography and mass spectrometry; 3HD, 3-hydroxydecanoic acid; 3HPTB, 3-hydroxypropylthiobutyric acid; 3HPTHx, 3-hydroxypropylthiohexanoic acid; 3HPTO, 3-hydroxypropylthiooctanoic acid; PHA, polyhydroxyalkanoate; PHAMCL, medium-chain-length polyhydroxyalkanoate; poly(3HPTA), poly(3-hydroxy-S-propyl--thioalkanoate); PTO, propylthiooctanoic acid; PTUD, propylthioundecanoic acid
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INTRODUCTION |
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Interestingly, some PHA synthases, such as the enzyme from Ralstonia eutropha, also accept mercaptoalkyl-CoA thioesters, such as 3-mercaptopropionyl-CoA or 3-mercaptobutyryl-CoA, as substrates, thus allowing the synthesis of polythioesters (PTE). PTEs, in which the constituents are covalently linked by thioester linkages, were only recently described and they represent a new (the eighth class) of biopolymers (Lütke-Eversloh et al., 2001a , b
).
Sulfur-containing biopolymers are rare and, besides those proteins that contain methionine and cysteine, some complex polysaccharides that contain sulfate groups and the PTEs, sulfur has to the best of our knowledge so far been identified only once in PHAs, in PHAs that contain 3-hydroxy-5-thiophenoxyalkanoic acids (Takagi et al., 1999 ). The aim of this study was to identify and metabolically engineer bacteria that synthesize PHAs with constituents containing aliphatic side chains with thioether linkages. The study was also done to shed some light on the catabolism of sulfur-containing fatty acids with respect to PHA biosynthesis in bacteria, because the catabolism of these sulfur compounds has been scarcely investigated in prokaryotic micro-organisms (Skrede et al., 1997
). Such PHAs have not been described before, and they will possibly attract much interest because they are accessible to chemical reactions allowing modifications and cross-linking of the polymer chains.
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METHODS |
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Cultivation of bacteria.
Organisms were cultured in nutrient broth (NB) or in mineral salts medium (MSM) according to Schlegel et al. (1961) . Different sodium salts of alkylthioalkanoic acids were added to these media as carbon sources, from filter-sterilized 10% (w/v) aqueous stock solutions at the concentrations indicated in the text. Fed-batch cultures were grown under aerobic conditions at 30 °C in 2 l Erlenmeyer flasks containing 500 ml of the appropriate medium and were agitated at 130 r.p.m. To promote PHA accumulation, the ammonia concentration was reduced to 0·05% (w/v) in MSM.
Fed-batch cultivation of R. eutropha PHB-4(pBBR1::phaC1) was done at a 26-litre scale in a stirred (at 50400 r.p.m.) and aerated (1520 l min-1) 30 l stainless-steel Biostat UD30 fermenter (B. Braun, Biotech International). The fermentation was carried out in MSM. The temperature (30 °C) and pH (7·0) were automatically controlled; the airflow rate and the stirring speed were 1·0 v.v.m. [gassing vol. (culture vol.)-1 min-1] and 400 r.p.m., respectively. Cell growth was monitored spectrophotometrically at 600 and 850 nm. The MSM contained 1·0% (w/v) sodium gluconate and 300 mg kanamycin l-1 from the beginning. Sodium gluconate and ammonium chloride were fed additionally, according to the respective growth parameters. Propylthiooctanoic acid was successively added at the end of the exponential growth phase in portions of 0·05% (w/v) from a filter-sterilized stock solution (10%, w/v) at intervals of 1 or 2 h. The final concentration of propylthiooctanoic acid (PTO) was 0·8% (w/v). At the end of the cultivation experiment, the cells were harvested by centrifugation and lyophilized.
Chemical synthesis of alkylthioalkanoic acids.
Propylthioundecanoic acid (PTUD), PTO, propylthiohexanoic acid, propylthiobutyric acid, propylthiopropionic acid, butylthiovaleric acid (BTV) and octylthiohexanoic acid were chemically synthesized from alkylthiol and -bromoalkanoic acids (Fig. 1
), according to the protocol of Skrede et al. (1997)
. The products were identified by gas chromatography and mass spectrometry (GC/MS). Propanethiol, butanethiol, octanethiol, 11-bromoundecanoic acid, 8-bromooctanoic acid, 6-bromohexanoic acid, 5-bromovaleric acid, 4-bromobutyric acid and 3-bromopropionic acid were purchased from Sigma.
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GC/MS analysis.
The polymer content and composition were determined by methanolysis of 57 mg of lyophilized cells or purified polymer in the presence of sulfuric acid; the resulting methyl esters were characterized by gas chromatography (Brandl et al., 1988 ).
According to the lability of thioether compounds to acid-catalysed methylation, esterification of purified polymer samples was accomplished by using (trimethylsilyl-)diazomethane as the methylating agent (Preu, 1999 ).
The identification of the compounds was achieved by GC/MS (HP 6890/HP 5973, Hewlett Packard).
Partial pyrolysis of polymers.
Identification of the constituents of PHAs was also performed by partial pyrolysis. For this, 5 mg of isolated polymer were transferred to the bottom of a small glass tube, which was subsequently flushed with argon and evacuated to 1 mbar. The lower part of the tube was then heated to approximately 500 °C until the polymer was completely degraded. Decomposition products condensed in the upper part of the glass tube. After cooling to room temperature, the products were dissolved in 2 ml methanol/chloroform (1:1). One part of this solution was used as obtained for electrospray-ionization/mass spectrometry (ESI-MS), and the other part was esterified with (trimethylsilyl)diazomethane for GC/MS.
ESI-MS and ESI-MS/MS analysis.
This soft ionization method was used to characterize the oligomers formed by pyrolysis of the polymers. All measurements were done on a Quattro LCZ (Micromass) apparatus with nanospray inlet.
Elemental sulfur analysis.
This 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 the purified polyesters were estimated by gel-permeation chromatography (GPC) relative to polystyrene standards (85, 120, 198, 280, 500, 810 and 990 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 , respectively) connected in line in a GPC apparatus (Waters). Samples were eluted with chloroform at a flow rate of 1·0 ml min-1 and at 35 °C; the eluted compounds were monitored using a Waters 410 differential refractometer. Polydispersity and the number mean (MN) and weight mean (MW) molar masses were calculated by using the Millenium Chromatography Manager GPC software (Waters).
Infrared spectroscopic analysis.
The infrared spectra were taken with a fourier transform spectrometer IFS 28 (Bruker). The samples were dissolved in CHCl3 and deposited as a film on a NaCl disk.
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RESULTS |
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GC analyses of cells of P. mendocina, P. aeruginosa and P. oleovorans did not provide evidence that the cells had synthesized PHAs containing unusual constituents after cultivation in the presence of ATAs, even if various co-substrates were added to the medium. The medium-chain-length PHA (PHAMCL) accumulated by these bacteria consisted mainly of 3-hydroxyhexanoic acid, 3-hydroxyheptanoic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid (3HD) and 3-hydroxydodecanoic acid, depending on the cultivation conditions.
PHA accumulation of P. putida KT2440 in the presence of PTUD
The growth of P. putida KT2440 was significantly lower in comparison to the other pseudomonads when PTO or PTUD was provided as sole carbon source. Interestingly, P. putida KT2440 was the only Pseudomonas species investigated in this study for which GC analysis revealed an unusual composition of PHAs after cultivation in MSM containing 0·35% (w/v) PTUD and equimolar amounts of nonanoic acid (v/v). Since it was suspected that the accumulated PHA contained thioether functional groups, an elemental sulfur analysis was done for the isolated polymer. It revealed a content of 6·02% (w/w) sulfur. Besides 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, and small amounts of 3-hydroxyoctanoic acid, 3HD, 3-hydroxyundecanoic acid and 3-hydroxydodecanoic acid, a significant amount of different sulfur-containing constituents was detected by gas chromatography. However, due to the complex composition, the analyses of the partially pyrolysed polymer did not exhibit information on the exact chemical structure of these putative novel polymer constituents.
Biosynthesis of poly(3-hydroxy-S-propyl--thioalkanoate) [poly(3HPTA)] from PTO by a recombinant strain of R. eutropha
We used a genetically engineered strain of the PHA-negative mutant PHB-4 of R. eutropha harbouring plasmid pBBR1::phaC1. This strain expressed the PHAMCL synthase from P. mendocina, and was used for further growth and PHA-accumulation studies in which ATAs were provided as carbon sources.
Since growth of R. eutropha PHB-4(pBBR1::phaC1) in MSM containing PTO as sole carbon source was very poor, cultivations were carried out in complex medium (NB) or in MSM containing sodium gluconate as a second utilizable carbon source, in addition to PTO. In the presence of PTO in the medium, R. eutropha PHB-4(pBBR1::phaC1) synthesized a hitherto unknown polyester, revealing exclusively 3-hydroxypropylthiobutyric acid, 3-hydroxypropylthiohexanoic acid and 3-hydroxypropylthiooctanoic acid as polymer constituents [poly(3HPTB-co-3HPTHx-co-3HPTO)] (see below). The cultivation conditions and the PHA contents of the cells, as well as the sulfur contents, molecular masses and polydispersity indices of the isolated poly(3HPTA)s obtained from four different cell batches, are shown in Table 1.
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Employing the established and widely used gas chromatographic method for PHA analysis (Brandl et al., 1988 ), the identification of the 3HPTA methyl esters failed, because these polymer constituents were sensitive to hot acid methanolysis conditions due to the thioether groups. Therefore, other methods were chosen to analyse the polymer composition.
After pyrolysis of the polymer and subsequent methylation of the pyrolysis mixture with (trimethylsilyl-)diazomethane, three different monomeric S-propyl--thioalkenoic acids were identified by GC/MS (Fig. 4
). The double bond was formed by the elimination of the adjacent fatty acid during the decomposition process. The double bond may be located either between the
- and ß-atom or between the ß- and
-atom. Due to the conjugated
-electron system, a double bond between the
- and ß-atom is most likely. The masses of the two compounds would not differ.
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Because GC analysis gave only information about the monomers, soft ionization techniques with nanospray inlet were used to identify a larger selection of sub-structures. Negative-ion ESI-MS (Fig. 5) showed groups of oligomers formed by combinations of 3HPTB, 3HPTHx and 3HPTO. ESI-MS/MS of the pseudomolecular ions suggested that the monomers were randomly combined (Table 2
). The fragmentation of the trimer m/z 535 is given as an example (Fig. 6
). The loss of the three possible monomers (as their corresponding olefins) with similar intensities gave proof that the trimer was a mixture of isomers with 3HPTB-, 3HPTHx- and 3HPTO-terminations.
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Biosynthesis of poly(3HPTA) from propylthiohexanoic acid by the recombinant strain of R. eutropha
The recombinant mutant PHB-4 of R. eutropha, expressing the P. mendocina PHA synthase, was also cultivated on propylthiohexanoic acid under the same conditions as described above for the cultivations on PTO. The accumulated polymer was isolated and subjected to chemical analysis as described above. Infrared and NMR spectroscopy, and GC and GC/MS analysis, as well as pyrolysis of the purified polymer, revealed that the polyester consisted of 3HPTB and 3HPTHx as sole constituents. Upon repeating similar cultivation experiments three times with propylthiobutyric acid, the cells did not accumulate any polyester that could be detected or isolated.
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DISCUSSION |
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The PHA-negative strain of R. eutropha PHB-4 was not able to synthesize poly(3HPTB-co-3HPTHx-co-3HPTO), thus confirming that a PHA synthase is involved in the synthesis of the novel PHAs. Since this strain is also not able to synthesize any other PHAs, a recombinant strain of PHB-4 expressing the type-II PHA synthase of P. mendocina, which exhibited a similar structure and broad substrate range to PHAMCL synthases of other pseudomonads (Hein et al., 2002 ; Rehm & Steinbüchel, 1999
), was chosen as a candidate for the production of these novel PHAs. The broad substrate range of PHA synthases and in particular that of type-II PHA synthases for substrates of different carbon chain length, which also allows the incorporation of hydroxyalkanoic acids containing various substituents at the alkyl moiety, is well known (Steinbüchel & Valentin, 1995
).
Interestingly, a smell characteristic for alkylthiols occurred if pseudomonads were cultivated with ATAs that contained an odd number of carbon atoms in the carboxylic acid part of the molecule, such as PTUD or BTV. This indicates a spontaneously occurring cleavage of the 3-hydroxy-S-alkyl--thiopropionyl-CoA into malonic acid semialdehyde CoA and the alkylthiol, whose further metabolic fate remains to be elucidated (Skrede et al., 1997
). In contrast, ATAs containing even numbers of carbon atoms in the carboxylic acid part of the molecule are presumably catabolized via the ß-oxidation route to acetyl-CoA until the presence of the sulfur atom prevents another ß-oxidation cycle. According to Skrede et al. (1997)
, the sulfur is metabolized to a sulfoxide, and further oxidation from the
-end is most likely to occur. A scheme for the putative metabolic pathway of ATA degradation and for poly(3HPTA) biosynthesis is presented in Fig. 8
.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 26 October 2001;
revised 18 January 2002;
accepted 22 January 2002.
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