Polyhydroxyalkanoate (PHA) Synthases: The Key Enzymes of PHA Synthesis

Rehm, Bernd H. A.; Steinbüchel, Alexander

doi:10.1002/3527600035.bpol3a06

Cited by 32 publications

(40 citation statements)

References 117 publications

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“…One of the most abundant polyhydroxyalkanoates is poly(3-hydroxybutyrate) (PHB), a homopolymer of D-(Ϫ)-3-hydroxybutyrate (3HB). PHB is synthesized from D-(Ϫ)-3-hydroxybutyryl-coenzyme A by PHB synthase (16,18) and is mobilized by intracellular PHB depolymerases (15,19).PHB-producing bacteria contain intracellular PHB depolymerases. Intracellular PHB depolymerases have been found in Rhodospirillum rubrum (15), Paracoccus denitrificans (4), and Wautersia eutropha (19) (formerly Ralstonia eutropha).…”

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Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

et al. 2005

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Wautersia eutropha H16 (formerly Ralstonia eutropha) mobilizes intracellularly accumulated poly(3-hydroxybutyrate) (PHB) with intracellular poly(3-hydroxybutyrate) depolymerases. In this study, a novel intracellular 3-hydroxybutyrate-oligomer hydrolase (PhaZc) gene was cloned and overexpressed in Escherichia coli. Then PhaZc was purified and characterized. Immunoblot analysis with polyclonal antiserum against PhaZc revealed that most PhaZc is present in the cytosolic fraction and a small amount is present in the poly(3-hydroxybutyrate) inclusion bodies of W. eutropha. PhaZc degraded various 3-hydroxybutyrate oligomers at a high specific activity and artificial amorphous poly(3-hydroxybutyrate) at a lower specific activity. Native PHB granules and semicrystalline PHB were not degraded by PhaZc. A PhaZ deletion mutation enhanced the deposition of PHB in the logarithmic phase in nutrient-rich medium. PhaZc differs from the hydrolases of W. eutropha previously reported and is a novel type of intracellular 3-hydroxybutyrate-oligomer hydrolase, and it participates in the mobilization of PHB along with other hydrolases.Polyhydroxyalkanoates, which are bacterial polyesters, are a group of storage materials that are used as sources of carbon and energy (1). One of the most abundant polyhydroxyalkanoates is poly(3-hydroxybutyrate) (PHB), a homopolymer of D-(Ϫ)-3-hydroxybutyrate (3HB). PHB is synthesized from D-(Ϫ)-3-hydroxybutyryl-coenzyme A by PHB synthase (16,18) and is mobilized by intracellular PHB depolymerases (15,19).PHB-producing bacteria contain intracellular PHB depolymerases. Intracellular PHB depolymerases have been found in Rhodospirillum rubrum (15), Paracoccus denitrificans (4), and Wautersia eutropha (19) (formerly Ralstonia eutropha). In W. eutropha H16, a gene encoding an intracellular PHB depolymerases, phaZ1 (19), and a gene encoding a 3HB-oligomer hydrolase, phaZb (formerly phaZ2) (10,20), have been cloned, and some properties of their products have been reported. Both hydrolases showed hydrolytic activity for amorphous PHB. PhaZb hydrolyzed 3HB oligomers at a high specific activity and degraded PHB into 3HB monomers at a lower specific activity.The function of PhaZ1 or PhaZb in vivo has been examined by constructing a phaZ1 or phaZb deletion strain (5, 10, 19). The deletion mutants exhibit considerably less degradation of PHB in vivo in some conditions. However, even a PhaZ1-PhaZb double-deletion mutation did not completely inhibit the mobilization of PHB (10). Therefore, it has been suggested that there are other depolymerases or related hydrolases (10).Since the amino acid sequence of the first intracellular PHB depolymerase, PhaZ1, was reported for W. eutropha H16, several isoenzymes have been found. Recently, the genome sequences of W. eutropha and related bacteria have become available. Four putative PHB depolymerase genes homologous to phaZ1 have been identified using the genome sequence of Wautersia metallidurans (32), the sequence of the megaplasmid of W. eutropha (25), and the incomplete gen...

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Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

et al. 2005

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“…In addition, PHA synthases are also capable of biosynthesis of polythioesters, which are in contrast to polyoxoesters composed of mercaptoalkanoic acids (23,24). At present, all known PHA synthases can be assigned to one of four different classes according to their substrate ranges and their subunit compositions (32). PHAs have attracted much interest because they are biodegradable thermoplastics or elastomers which could be used for many applications in industry, medicine, pharmacy, and other areas (see references 1 and 6 for reviews).…”

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Polyhydroxyalkanoate (PHA) Accumulation in Sulfate-Reducing Bacteria and Identification of a Class III PHA Synthase (PhaEC) in Desulfococcus multivorans

Hải

Lange

Rabus

et al. 2004

Appl Environ Microbiol

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Seven strains of sulfate-reducing bacteria (SRB) were tested for the accumulation of polyhydroxyalkanoates (PHAs). During growth with benzoate Desulfonema magnum accumulated large amounts of poly(3-hydroxybutyrate) [poly(3HB)]. Desulfosarcina variabilis (during growth with benzoate), Desulfobotulus sapovorans (during growth with caproate), and Desulfobacterium autotrophicum (during growth with caproate) accumulated poly(3HB) that accounted for 20 to 43% of cell dry matter. Desulfobotulus sapovorans and Desulfobacterium autotrophicum also synthesized copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyvalerate when valerate was used as the growth substrate. Desulfovibrio vulgaris and Desulfotalea psychrophila were the only SRB tested in which PHAs were not detected. When total DNA isolated from Desulfococcus multivorans and specific primers deduced from highly conserved regions of known PHA synthases (PhaC) were used, a PCR product homologous to the central region of class III PHA synthases was obtained. The complete pha locus of Desulfococcus multivorans was subsequently obtained by inverse PCR, and it contained adjacent phaE Dm and phaC Dm genes. PhaC Dm and PhaE Dm were composed of 371 and 306 amino acid residues and showed up to 49 or 23% amino acid identity to the corresponding subunits of other class III PHA synthases. Constructs of phaC Dm alone (pBBRMCS-2::phaC Dm ) and of phaE Dm C Dm (pBBRMCS-2::phaE Dm C Dm ) in various vectors were obtained and transferred to several strains of Escherichia coli, as well as to the PHA-negative mutants PHB ؊ 4 and GPp104 of Ralstonia eutropha and Pseudomonas putida, respectively. In cells of the recombinant strains harboring phaE Dm C Dm small but significant amounts (up to 1.7% of cell dry matter) of poly(3HB) and of PHA synthase activity (up to 1.5 U/mg protein) were detected. This indicated that the cloned genes encode functionally active proteins. Hybrid synthases consisting of PhaC Dm and PhaE of Thiococcus pfennigii or Synechocystis sp. strain PCC 6308 were also constructed and were shown to be functionally active.Sulfate-reducing bacteria (SRB) are anaerobic bacteria that couple the oxidation of simple organic compounds (e.g., lactate) or of molecular hydrogen to the reduction of sulfate, which is the dominant electron acceptor in anaerobic zones of marine ecosystems, yielding the conspicuous end product hydrogen sulfide. SRB contribute significantly to the global cycling of carbon and sulfur, mineralizing more than 50% of the organic carbon via sulfate reduction in marine sediments (15,16). In addition, they also occur in brackish and freshwater habitats. SRB represent different genera and species, most of which are affiliated with the ␦ subgroup of the Proteobacteria, and they display a diversity of nutritional and catabolic properties (30,45,47). Besides their ecological role in carbon and sulfur cycles, SRB are also economically relevant, since they are implicated in corrosion of iron, concrete, and other materials (26). Despite a strong interest in the...

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“…Intensive research has been carried out on the physiology, biochemistry, and molecular genetics of the metabolism of PHAs in various naturally PHA-producing bacteria during the last two decades (26,33). High-cell-density culture techniques for culturing recombinant strains of Escherichia coli were developed for production of various PHAs to reduce the production cost and to increase productivity (9,10,11,16,27).…”

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Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli , Including a Novel In Situ Isolation Method

Thakor

Lütke‐Eversloh

Steinbüchel

2005

Appl Environ Microbiol

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Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30-and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90°C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.

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Polyhydroxyalkanoate (PHA) Synthases: The Key Enzymes of PHA Synthesis

Abstract: Introduction Terminology Cloning Strategies for PHA Synthase Genes Organization of PHA Synthase Genes Primary Structures of PHA Synthases Secondary and Quaternary Structures of PHA Synthases In vivo versus in vitro Substrate Specificity of PHA Synthases… Show more

Cited by 32 publications

References 117 publications

Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Novel Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase in Wautersia eutropha H16

Polyhydroxyalkanoate (PHA) Accumulation in Sulfate-Reducing Bacteria and Identification of a Class III PHA Synthase (PhaEC) in Desulfococcus multivorans

Application of the BPEC Pathway for Large-Scale Biotechnological Production of Poly(3-Mercaptopropionate) by Recombinant Escherichia coli , Including a Novel In Situ Isolation Method

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