In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

Qi, Qungang; Steinbüchel, Alexander; Rehm, Bernd H. A.

doi:10.1007/s002530000357

Cited by 95 publications

(68 citation statements)

References 19 publications

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“…These data suggested that the fatty acid de novo synthesis as well as the ␤-oxidation pathways were involved. It was recently confirmed that the purified PHA MCL synthases from P. aeruginosa exhibit in vitro enzyme activity with (R)-3-hydroxydecanoyl-CoA as the substrate (26). Thus, to serve as a substrate for the PHA synthase, (R)-3-hydroxyacyl-acyl carrier protein [(R)-3-hydroxyacyl-ACP], which is an intermediate of fatty acid de novo synthesis, must be converted to the corresponding CoA-derivative.…”

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confidence: 92%

Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli

Rehm

Mitsky

Steinbüchel

2001

Appl Environ Microbiol

140

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Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the ␤-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.

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confidence: 92%

Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli

Rehm

Mitsky

Steinbüchel

2001

Appl Environ Microbiol

140

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“…The 1.3-kb BamHI-HindIII fragment containing the P. putida phaG gene was isolated from plasmid pBHR75 (14) and subcloned into the respective sites of plasmid pBHR71 (8). The resulting plasmid, pBHR73, was hydrolyzed with XbaI, and a fill-in reaction was performed with the large fragment of DNA polymerase I.…”

Section: Methodsmentioning

confidence: 99%

PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Fiedler

Steinbüchel

Rehm

2000

Appl Environ Microbiol

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Recently, a new metabolic link between fatty acid de novo biosynthesis and biosynthesis of poly(3-hydroxyalkanoate) consisting of medium-chain-length constituents (C 6 to C 14 ) (PHA MCL ), catalyzed by the 3-hydroxydecanoyl-[acyl-carrier-protein]:CoA transacylase (PhaG), has been identified in Pseudomonas putida (B. H. A. Rehm, N. Krüger, and A. Steinbüchel, J. Biol. Chem. 273:24044-24051, 1998). To establish this PHA-biosynthetic pathway in a non-PHA-accumulating bacterium, we functionally coexpressed phaC1 (encoding PHA synthase 1) from Pseudomonas aeruginosa and phaG (encoding the transacylase) from P. putida in Pseudomonas fragi. The recombinant strains of P. fragi were cultivated on gluconate as the sole carbon source, and PHA accumulation to about 14% of the total cellular dry weight was achieved. The respective polyester was isolated, and GPC analysis revealed a weight average molar mass of about 130,000 g mol ؊1 and a polydispersity of 2.2. The PHA was composed mainly (60 mol%) of 3-hydroxydecanoate. These data strongly suggested that functional expression of phaC1 and phaG established a new pathway for PHA MCL biosynthesis from nonrelated carbon sources in P. fragi. When fatty acids were used as the carbon source, no PHA accumulation was observed in PHA synthase-expressing P. fragi, whereas application of the ␤-oxidation inhibitor acrylic acid mediated PHA MCL accumulation. The substrate for the PHA synthase PhaC1 is therefore presumably directly provided through the enzymatic activity of the transacylase PhaG by the conversion of (R)-3-hydroxydecanoyl-ACP to (R)-3-hydroxydecanoyl-CoA when the organism is cultivated on gluconate. Here we demonstrate for the first time the establishment of PHA MCL synthesis from nonrelated carbon sources in a non-PHA-accumulating bacterium, employing fatty acid de novo biosynthesis and the enzymes PhaG (a transacylase) and PhaC1 (a PHA synthase).Most fluorescent pseudomonads belonging to rRNA homology group I are able to synthesize and accumulate large amounts of polyhydroxyalkanoic acids (PHAs) consisting of various 3-hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (medium chain length [MCL]) as carbon and energy storage compounds (1,22). Pseudomonas fragi is an exception; it is not able to accumulate PHA from either fatty acids or other simple carbon sources such as gluconate (26). PHA composition depends on the PHA synthases present, the carbon source, and the metabolic routes involved (9,16,21). In Pseudomonas putida there are at least three different metabolic routes for the synthesis of 3-hydroxyacyl coenzyme A (CoA) thioesters, which are the substrates of PHA synthase (4, 15). ␤-Oxidation is the main pathway when fatty acids are used as the carbon source. Fatty acid de novo biosynthesis is the main route during growth on a carbon source which is metabolized to acetyl-CoA, like gluconate, acetate, or ethanol. The chain elongation reaction, in which acetyl-CoA moieties are condensed to 3-hydroxyacyl-CoA, is involved in PHA synthesis ...

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“…Purified PHA synthase and suitable CoA-activated monomers in an aqueous buffer are sufficient for in vitro synthesis of PHA (17,39,41,78,91). If the metabolism of a given species is able to provide suitable precursor substrates (acyl-CoAs), recombinantly expressed PHA synthase catalyzes the formation of PHA in vivo in different host such as plants, animals, or yeasts (49,72,120).…”

Section: Pha Synthasesmentioning

confidence: 99%

Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes)

2009

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Polyhydroxyalkanoates (PHAs) such as poly(3-hydroxybutyrate) (PHB) or poly(3-hydroxyoctanoate), are universal prokaryotic storage compounds of carbon and energy. PHAs are accumulated intracellularly in form of inclusion bodies (PHA granules) during times of oversupply with carbon sources (for reviews, see references 2, 54, 64, 76, 86, and 100). PHAs can consist of short-chain-length hydroxyalkanoic acids (PHA SCL ) or medium-chain-length monomers (PHA MCL ), depending on strain and culture conditions. Given a number of more than 150 identified hydroxyalkanoates as potential constituents (101) the theoretical number of different PHA copolymers is incredibly high. A few PHAs, such as PHB and copolymers of 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and/or 4-hydroxybutyrate, are produced by industry (Biocycle, Biomer, Biopol, Enmat, Mirel, and Nodax).The number of publications on PHA metabolism has considerably increased in the last two decades, and many aspects of the biosynthesis, molecular architecture, intracellular mobilization, extracellular degradation, and commercial applications of PHAs have been addressed. One exiting outcome of these studies was the finding that many polypeptides are specifically present on the surface of PHA granules, much more than would be essential for PHA synthesis. These proteins constitute a particular surface layer on PHA granules that is essential for PHA metabolism. In conclusion, PHA granules are complexly organized subcellular structures and appear to be more than simple polymer inclusions. The current knowledge of the biochemical functions of PHA-associated proteins will be reviewed here. This will be done using the example of Ralstonia eutropha H16, which is the model organism of PHA research and has become an important prokaryotic strain for several biotechnological applications (82). It should be noted that several other taxonomic names of R. eutropha are found in literature. These include Hydrogenomonas eutropha, Alcaligenes eutrophus, Wautersia eutropha, and Cupriavidus necator. We used here the most commonly accepted name, R. eutropha, which has been also used for description of the recently sequenced genome (71). When appropriate, the properties of PHA-bound proteins of other organisms are discussed afterward. PHA SYNTHASESPHA synthase is the key enzyme of PHA synthesis and catalyzes the polymerization of hydroxyacyl-coenzyme A (CoA) to PHA and free CoA. The first PHA synthase gene (phaC) was cloned 20 years ago. Remarkably, this was done independently in three labs with the same bacterial strain, R. eutrophus H16 (66, 95, 97). Meanwhile, several PHA synthases from different sources have been biochemically investigated, and many more PHA synthase genes have been cloned or have been determined by genome sequencing. PHA synthases currently are divided into four classes depending on their subunit composition and substrate specificity. The R. eutropha and Pseudomonas putida (previously Pseudomonas oleovorans) PHA synthases represent class I (producing PHA SCL ) and class ...

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In vitro synthesis of poly(3-hydroxydecanoate): purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

Cited by 95 publications

References 19 publications

Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli

Role of Fatty Acid De Novo Biosynthesis in Polyhydroxyalkanoic Acid (PHA) and Rhamnolipid Synthesis by Pseudomonads: Establishment of the Transacylase (PhaG)-Mediated Pathway for PHA Biosynthesis in Escherichia coli

PhaG-Mediated Synthesis of Poly(3-Hydroxyalkanoates) Consisting of Medium-Chain-Length Constituents from Nonrelated Carbon Sources in Recombinant Pseudomonas fragi

Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes)

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