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

Abstract

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 ...

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs

“…Rehm et al (1998) showed that the metabolic link between FAB and mcl-PHA biosynthesis is accomplished by the action of ACP-CoA-transacylase, the phaG gene product. This enzyme is able to direct precursors from FAB to mclPHAs in several prokaryotic hosts (Rehm et al 1998;Fiedler et al 2000;Hoffmann et al 2000aHoffmann et al , 2000b. In the present report, a eukaryote, i.e.…”

“…The presence of unsaturated monomers in Pseudomonas oleovorans, P. fragi and P. putida KT2442 results from the contribution of boxidation to PHA biosynthesis. % PHAs, mg of polymer/100 mg of dry mass; H6, 3-(R)-hydroxyhexanoic acid; H8, 3-(R)-hydroxyoctanoic acid; H8:1, 3-(R)-hydroxyoctenoic acid; H10, 3-(R)-hydroxydecanoic acid; H12, 3-(R)-hydroxydodecanoic acid; H12:1, 3-(R)-hydroxydodecenoic acid; H12:2, 3-(R)-hydroxydodecandienoic acid; H14, 3-(R)-hydroxytetradecanoic acid; H14:1, 3-(R)-hydroxytetradecenoic;nd, not detectable; -, not determined (Hoffmann et al 2000b) b Strain not-accumulating mcl-PHAs from FAB, harbouring the phaG gene from P. putida KT2440 and grown on gluconate (Rehm et al 1998) c P. putida phaG negative mutant harbouring an active phaG gene from the wild-type strain (P. putida KT2440) and grown on gluconate (Rehm et al 1998) d Strain not accumulating mcl-PHAs from FAB, harbouring an active phaG from P. putida KT2440 and grown on gluconate (Fiedler et al 2000) e Untransformed strain grown on gluconate (Rehm et al 1998) f Untransformed strain grown on gluconate (Hoffmann et al 2000a) g Untransformed strain grown on glycerol (Huijberts et al 1992) 295-amino-acid polypeptide with a mass of 33.8 kDa. The protein shares high homology with other cloned phaG genes.…”

“…This enzyme catalyses the transfer of the 3-hydroxyacyl moiety from ACP to CoA (Rehm et al 1998). Expression of the phaG gene in a number of recombinant prokaryotic hosts led to the accumulation of mcl-PHAs from FAB (Fiedler et al 2000;Hoffmann et al 2000a).…”

Acyltransferases in Bacteria

“…The PhaG-mediated biosynthesis of PHA MCL from unrelated carbon sources appears to be a widespread strategy among various pseudomonads, but several species are not able to exploit this route. For example, P. fragi and P. jessenii are known to synthesize only very small amounts of PHA from sugars (287,288). A BLAST search reveals that their genomes do not encode a protein similar to PhaG.…”

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

“…Interestingly, in P. aeruginosa about 40% of the accumulated PHA is provided via alternative pathways from gluconate as the carbon source independent of the transacylase PhaG (14). In non-PHA-accumulating Pseudomonas fragi the coexpression of phaG Pp with phaC1 Pa established a new pathway for PHA MCL biosynthesis from fatty acid de novo biosynthesis using nonrelated carbon sources, e.g., gluconate or acetate (8).…”