Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany1
Author for correspondence: Bernd H. A. Rehm. Tel: +49 251 833 9848. Fax: +49 251 833 8388. e-mail: rehm{at}uni-muenster.de
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ABSTRACT |
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Keywords: polyhydroxyalkanoate, PHA synthase, PHA depolymerase
Abbreviations: CDW, cell dry weight; GCMS, gas chromatographymass spectrometry; GPC, gel permeation chromatography; PHA, polyhydroxyalkanoate; PHB, polyhydroxybutyrate
The GenBank accession numbers for the phaC and phaZ sequences reported in this paper are AY007313 and AF311864, respectively.
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INTRODUCTION |
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PHAs are currently under intensive investigation because of their inherent property as biodegradable thermoplastics. PHA synthases, which use CoA thioesters of (R)-3-hydroxyalkanoates as substrates and catalyse the polymerization of these monomers to PHA with concomitant release of CoA, represent the key enzymes of PHA biosynthesis. More than 40 PHA synthase genes have been assigned and characterized (Rehm & Steinbüchel, 1999 , 2001a
), and their protein products can be broadly arranged into three different classes based on their subunit composition and substrate specificities. Class I synthases are active towards short-chain-length (R)-hydroxyacyl-CoA, consisting of three to five carbon atoms, and are represented by the PHA synthase of Ralstonia eutropha. Class II is represented by the PHA synthase of Pseudomonas aeruginosa which is active towards medium-chain-length (R)-3-hydroxyacyl-CoA, containing 6 to 14 C atoms. PHA synthases of class I and class II are composed of a single subunit, whereas class III PHA synthases are composed of two non-identical subunits. Class III is represented by the Allochromatium vinosum PHA synthase, consisting of subunits PhaC and PhaE, and exhibiting activity towards short-chain-length (R)-3-hydroxyacyl-CoA. In addition, a few bacteria, such as Aeromonas punctata (Fukui et al., 1998
) and Rhodococcus ruber (Haywood et al., 1991
), have been reported to have PHA synthases that are composed of one subunit which exhibits specificity for both short- and medium-chain-length (R)-3-hydroxyacyl-CoA. Although numerous PHA synthase genes have been cloned and assigned, only a few PHA synthases have been purified and enzymically characterized, e.g. PHA synthases from R. eutropha, A. vinosum and P. aeruginosa (Gerngross et al., 1994
; Liebergesell et al., 1994
; Qi et al., 2000
; Rehm et al., 2001a
).
The PHB biosynthesis pathway of R. eutropha has been studied in detail (Peoples & Sinskey, 1989a , b
; Schubert et al., 1988
). In this bacterium, the biosynthetic process is initiated by the condensation of two acetyl-CoA molecules to produce acetoacetyl-CoA which is catalysed by the enzyme ß-ketothiolase (EC 2.3.1.9; gene phbA). Acetoacetyl-CoA is then reduced to (R)-3-hydroxybutyryl-CoA by the NADPH-dependent acetoacetyl-CoA reductase (EC 1.1.1.36; gene phbB). These enzymes have also been found and studied in several other PHB-accumulating bacteria.
The synthesis of medium-chain-length PHAs relies mainly on the ß-oxidation pathway (Langenbach et al., 1997 ; Qi et al., 1997
, 1998
) when fatty acids are used as carbon source, or on fatty acid de novo synthesis when other non-related carbon sources, such as gluconate, were employed (Rehm et al., 1998
; Fiedler et al., 2000
; Hoffmann et al. 2000a
, 2000b
). Meanwhile, various medium-chain-length PHA biosynthetic pathways have been established in recombinant bacteria, which recruit intermediates of fatty acid metabolism (Rehm et al., 2001b
; Rehm & Steinbüchel, 2001b
).
In this study we describe the capability of C. crescentus DSM 4727T to produce PHB in the presence of excess carbon source. The PHB synthase gene and corresponding enzyme of C. crescentus DSM 4727T were characterized by heterologous functional expression in a PHB-negative mutant of R. eutropha and in Escherichia coli, and by analysis of the in vitro PHB synthase activity. Furthermore, evidence for the re-utilization of PHB by an intracellular PHB depolymerase was also obtained.
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METHODS |
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Analysis of the PHA in cells.
PHAs were analysed by GC. Cells were harvested by centrifugation at 10000 g for 15 min. The cells were then washed twice in saline solution (0·9%, w/v, NaCl) and lyophilized overnight. About 510 mg dry cells were subjected to methanolysis in the presence of 15% (v/v) sulfuric acid. The resulting methyl esters of the respective 3-hydroxyalkanoates were assayed by a GC system (Perkin-Elmer) equipped with a 0·5 µm Permphase PEG25 Mx capillary column as described by Brandl et al. (1988) .
Isolation of the PHA from lyophilized cells.
PHA was extracted from lyophilized cells using chloroform extraction in a Soxhlet apparatus and was subsequently precipitated with 10 vols ethanol. The precipitate was dissolved in chloroform and ethanol-precipitated again in order to obtain highly purified PHA.
Gas chromatographymass spectrometry (GCMS).
Purified polymer, prepared as described above, was dissolved in chloroform (5 mg PHA ml-1), and 3 µl was injected into a GCMS instrument (model 6890; Hewlett Packard). The column and temperature profile used for GC analysis were as described by Schubert et al. (1991) .
Gel permeation chromatography (GPC).
Molecular mass analysis was conducted with purified PHA, which was dissolved in chloroform (510 mg PHA ml-1) and introduced into a GPC system (Waters). The GPC system was equipped with Styragel Guard and a Styragel HR36 separation column. The eluted polymer was detected with a differential refractometer (model 410; Waters). A polystyrene molecular mass standard (Sigma) with a narrow range of polydispersity was employed for calibration of the system.
Protein content.
The protein content was obtained by the Bradford method, as described by Laemmli (1970) .
Electrophoresis of proteins.
SDS-PAGE (12·5%, w/v) was performed in a vertical slab gel electrophoresis apparatus, as described by Sambrook et al. (1989) .
Western immunoblot analysis.
Western blots were performed with a semidry Fastblot apparatus (Bio-Rad) as follows. Antiserum against the PHB synthase of R. eutropha (anti-PhaC) was raised. This antiserum was applied to a nitrocellulose membrane, and an alkaline phosphataseantibody conjugate (Sigma) was also applied to the membrane. Bound antibodies were detected using nitro blue tetrazolium chloride and the toluidine salt of 5-bromo-4-chloro-3-indolyl phosphate.
PHB synthase assay.
The in vitro activity of PHB synthase in crude extracts was assayed by the DTNB [5,5'-dithiobios-(2-nitrobenzoic acid)] method, as described by Valentin & Steinbüchel (1993) .
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RESULTS AND DISCUSSION |
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Time-course analysis of PHB production by C. crescentus DSM 4727T using glucose as a carbon source indicated growth-associated PHB production (Fig. 1). PHB accumulated simultaneously with cell growth, and reached its maximum level after approximately 60 h (Fig. 1
), contributing approximately 18·3% of cell dry weight (CDW) (biomass 2·5 g CDW l-1). A slight decrease in the level of CDW coincided with a small decrease in PHB content, which indicated the presence of an intracellular PHB depolymerase. Further evidence for an intracellular depolymerase was obtained by identification of a conserved hypothetical protein (Locus no. AAK22237; GI no. 13421381) within the genome sequence for C. crescentus [GenBank accession no. AE005698 (part 24 of 359); Nierman et. al, 2001
] whose deduced amino acid sequence showed approximately 45% similarity to the intracellular depolymerase of R. eutropha (Handrick et al., 2000
). This putative phaZ gene encodes a protein consisting of 375 aa with a molecular mass of 41·8 kDa.
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A putative lipase box GX(C/S)XG was found within the PHB synthase protein sequence, in which the serine essential for the active site of the lipase was replaced by a cysteine. It also contained the conserved amino acids C319, D480 and H508 (positions relative to R. eutropha PHA synthase). Analysis of the adjacent DNA sequence regions (3 kb upstream and downstream of the respective coding region) did not indicate co-localization of PHB biosynthesis genes, such as phbA and phbB. We amplified and cloned the putative PHB-synthase-encoding DNA region as described in Methods. Plasmid pQQ4 was introduced into E. coli JM109 and R. eutropha PHB-4, and the cells were cultivated under the respective PHB accumulation conditions. SDS-PAGE and immunoblot analysis with anti-PhaC antiserum (raised against the R. eutropha protein) revealed that the putative PHB synthase gene was expressed in both E. coli JM109 and R. eutropha PHB-4 (Fig. 2). An immunologically cross-reacting protein of approximately 73 kDa was obtained. A cross-reacting protein with an apparent molecular mass of 73 kDa was also detected, by anti-PhaC antiserum in crude extracts of C. crescentus (Fig. 2
).
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Enzymic activity of the putative PHB synthase from C. crescentus
Crude extracts of C. crescentus, recombinant R. eutropha PHB-4(pQQ4) and recombinant E. coli JM109(pQQ4) were investigated with respect to in vitro PHB synthase activity, employing 3-hydroxybutyryl-CoA as substrate (Table 2). In C. crescentus, a PHB synthase activity of 59·5 mU (mg total protein)-1 was obtained. The PHB synthase activity in crude extracts of recombinant R. eutropha PHB-4(pQQ4) and recombinant E. coli JM109(pQQ4) was 304 mU (mg total protein)-1 and 54·5 mU (mg total protein)-1, respectively. As expected, no activity was detected for the control strains, carrying only vector pBBR1-JO2. Hence, the study indicated that the PHB synthase shows in vitro activity in C. crescentus and that the enzyme is functionally produced in R. eutropha PHB-4 and E. coli JM109.
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ACKNOWLEDGEMENTS |
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Received 11 June 2001;
revised 30 July 2001;
accepted 14 August 2001.
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