Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters

Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate.

Polyhydroxyalkanoates (PHAs) are biopolyesters composed of hydroxy fatty acids, which represent a complex class of storage polyesters. They are synthesized by a wide range of different Gram-positive and Gram-negative bacteria, as well as by some Archaea, and are deposited as insoluble cytoplasmic inclusions. Polyester synthases are the key enzymes of polyester biosynthesis and catalyse the conversion of (R)-hydroxyacyl-CoA thioesters to polyesters with the concomitant release of CoA. These soluble enzymes turn into amphipathic enzymes upon covalent catalysis of polyester-chain formation. A self-assembly process is initiated resulting in the formation of insoluble cytoplasmic inclusions with a phospholipid monolayer and covalently attached polyester synthases at the surface. Surface-attached polyester synthases show a marked increase in enzyme activity. These polyester synthases have only recently been biochemically characterized. An overview of these recent findings is provided. At present, 59 polyester synthase structural genes from 45 different bacteria have been cloned and the nucleotide sequences have been obtained. The multiple alignment of the primary structures of these polyester synthases show an overall identity of 8-96% with only eight strictly conserved amino acid residues. Polyester synthases can been assigned to four classes based on their substrate specificity and subunit composition. The current knowledge on the organization of the polyester synthase genes, and other genes encoding proteins related to PHA metabolism, is compiled. In addition, the primary structures of the 59 PHA synthases are aligned and analysed with respect to highly conserved amino acids, and biochemical features of polyester synthases are described. The proposed catalytic mechanism based on similarities to alpha/beta-hydrolases and mutational analysis is discussed. Different threading algorithms suggest that polyester synthases belong to the alpha/beta-hydrolase superfamily, with a conserved cysteine residue as catalytic nucleophile. This review provides a survey of the known biochemical features of these unique enzymes and their proposed catalytic mechanism.

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Polyester synthases: natural catalysts for plastics.

Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis

Bioplastics from microorganisms.

Polyhydroxyalkanoates (PHAs) are accumulated in various bacteria as intracellular carbon and energy storage material under nutrient-limited conditions (3, 26, 30) These bacterial PHAs are expected to become attractive alternatives for petrochemically based plastics, since they are biodegradable thermoplastics More than 90 different constituent monomer units have been found (47) The PHA-producing bacteria can be broadly divided into two groups according to the number of carbon atoms in the monomeric units of the PHAs produced (44) One group of bacteria, including Ralstonia eutropha (formerly Alcaligenes eutrophus), produces short chain length PHAs with C3 to C5 monomer units, while the other group, including Pseudomonas oleovorans, produces medium chain length PHAs with C6 to C14 monomer units (3, 43)

Although the majority of bacteria accumulate either short chain length PHA or medium chain length PHA, several bacteria have been found to synthesize polyesters containing both short and medium chain length 3-hydroxyalkanoic acids (3HA) The bacteria Rhodospirillum rubrum (4), Rhodocyclus gelatinosus (27), and Rhodococcus sp (13) produced terpolyesters consisting of 3HA units of C4, C5, and C6 from hexanoate Aeromonas caviae produced a random copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx) (6, 8, 38) Pseudomonas strain GP4BH1 produced PHA containing 3HB and 3-hydroxyoctanoate (3HO) from octanoate and PHA containing 3HB, 3HO, and 3-hydroxydecanoate (3HD) from gluconate (46) In this bacterium, a polymer blend was suggested to be synthesized rather than a copolymer A recombinant strain of P oleovorans expressing R eutropha poly(3HB) [P(3HB)] biosynthesis genes has been shown to synthesize a blend of a P(3HB) homopolymer and a copolymer of 3HHx and 3HO units when grown on octanoate (49) Both polyesters were stored as separated granules within the cells (32) In addition, Pseudomonas fluorescens and several other Pseudomonas strains were found to produce a poly(3HB-co-3HA) [P(3HB-co-3HA)] copolymer consisting of 3HA units of C4 to C12 from 3HB and 1,3-butanediol (25) Although Thiocapsa pfennigii accumulated only a P(3HB) homopolymer from various carbon sources, a recombinant P putida strain harboring the PHA synthesis genes of T pfennigii produced a P(3HB-co-3HHx-co-3HO) terpolymer from octanoate (28)

We have reported that Pseudomonas sp strain 61-3 isolated from soil produces a blend of a P(3HB) homopolymer and a random copolymer [P(3HB-co-3HA)] consisting of 3HA units of C4 to C12 from sugars and alkanoic acids (1, 19, 20) In addition, two different types of polyester granules were formed in the same cell (9, 21) This suggests that Pseudomonas sp strain 61-3 possesses two types of polyester synthases with different substrate specificities, that is, polyhydroxybutyrate (PHB) synthase and PHA synthase, specific for 3HB and 3HA units ranging from C4 to C12, respectively In this study, we cloned and sequenced the P(3HB) biosynthesis genes, as well as the P(3HB-co-3HA) biosynthesis genes, of Pseudomonas sp strain 61-3 The substrate specificity of each polyester synthase was evaluated by heterologous expression in PHA-negative mutants of P putida and R eutropha In addition, we found that the phbRPs gene product exhibits significant similarity to the AraC/XylS family of transcriptional activators and report that it is a positive regulatory protein that controls the expression of the P(3HB) biosynthesis operon

Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3.

Sumihide Manji

Papers

Cloning and molecular analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3.