P-Ketothiolase

In order to produce PHAs in plants it is necessary to introduce the biosynthetic enzymes from bacteria. PHB represents the best characterized and simplest form of PHA, and the synthetic pathway (Figure 4.2) has been extensively studied in Ralstonia eutropha. 30,31 Starting from acetyl-CoA, a P-ketothiolase is required in order to form acetoacetyl-CoA. This is then reduced by a NADPH-dependent acetoacetyl-CoA reductase, which gives rise to 3-hydroxybutyryl-CoA. The latter intermediate is the substrate for the polymerization reaction catalyzed by polyhydroxybutyrate synthase.30 In Ralstonia eutropha, the thiolase, reductase, and synthase genes make up an operon.31... 

The copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB-co-PHV) produced by A eutrophus has generated more interest than poly-(R)-3-hydroxybutyrate (PHB) homopolymer. Since these bacterial polyesters are biodegradable thermoplastics, their mechanical and physical properties have received much attention. PHB is a relatively stiff and brittle material because of its high crystallinity. However, the physiochemi-cal and mechanical properties of [P(HB-HV)] vary widely and depend on the molar percentage of 3-hydroxyvalerate (HV) in the copolymer (4,5) as shown inTable 1. Propionic acid is converted by a synthetase to propionyl-CoA, and the biosynthetic P-ketothiolase catalyzes the condensation of propionyl-CoA with acetyl-CoA to 3-ketovaleryl-CoA by the acetoacetyl-CoA reductase. The hydroxyvaleryl moiety is finally covalently linked to the polyester by the PHA synthase (6). 

Fig.l. Biologic and chemical processes for production of R3HB in natural and recombinant bacteria. PhaA,P-ketothiolase PhaB,acetoacetyl-CoA reductase PhaC, PHA synthase Ptb, phosphotransbutyrylase Buk, butyratekinase TCA, tricarboxylic acid. 

Acetoacetate is activated to acetoacetyl CoA by reacting with succinyl CoA. Acetoacetyl CoA is cleaved by P-ketothiolase to 2 acetyl CoAs, which enter the TCA cycle and are oxidized to C02 and H20, generating ATP. 

The phhA, phhB, and phbC genes from Alcaligenes eutrophus have been cloned into E. coli (Scheme 32). These genes encode for the biosynthetic enzymes P-ketothiolase, acetoacetyl-CoA reductase (NADPH-dependent), and PHB synthase, respectively [199-202]. These genes have been also cloned into plants, since the cost of the bacterially produced PHB was higher than other biomaterials such as starches or lipids [203,204]. 

CoA (Figure 36-3). This occurs only in liver mitochondria because of its critical role in gluconeogenesis. The ketone bodies are transported out of the liver mitochondria and the hver into the bloodstream for transport to other tissues where they reenter metabolism by being converted to acetoacetyl-CoA at the expense of succinyl-CoA and then cleaved by P-ketothiolase to produce two molecules of acetyl-CoA for metabolism in the TCA cycle. 

C. Leucine but none of the other amino acids listed is a branched-chain amino acid. The muscle has a very active branched-chain amino acid metabolic pathway and uses that pathway to provide energy for its own use. The products of leucine metabolism are acetyl-CoA and acetoacetate, which are used in the tricarboxylic acid cycle. Acetoacetate is activated by succinyl-CoA and cleaved to two molecules of acetyl-CoA in the P-ketothiolase reaction. The other branched-chain amino acids, valine, and isoleucine, yield succinyl-CoA and acetyl-CoA as products of their catabolism. 

AcylCoA dehydrogenase enoyl hydralote p-hydrox/ acylCoA dehydrogenase ifSi p-ketothiolase... 

The route for P(3HB) synthesis in C. necator is one of the simplest and extensively studied PHA biosynthetic pathway (Fig. 2.3). Via this route, P-ketothiolase (PhaA) condenses two molecules of acetyl-CoA to form ace-toacetyl-CoA. An NADH2-dependent acetoacetyl-CoA reductase (PhaB) then catalyzes the conversion of acetoacetyl-CoA to (f )-3-hydroxybutyryl-CoA. The PhaC catalyzes the polymerization of (/ )-3-hydroxybutyryl-CoA monomers into P(3HB) polymer (Sudesh et al. 2000 Khanna and Srivastava 2005a Suriyamongkol et al. 2007). 

In another study by Dennis and co-workers [41], the PHA accumulation of several microorganisms Escherichia coli, Klebsiella aerogenes and PHA-negative mutants of Cupriavidus necator and Pseudomonas putida) that expressed the phaC and acetoacetyl-coenzyme A reductase phaB) of Cupriavidus necator were analysed. This results in a construct that puts phaC and phaB under the control of the original Cupriavidus necator promoter. It was found that wild-type Cupriavidus necator was able to produce sc/-PHA, however the recombinant Cupriavidus necator expressing its own phaC and phaB without P-ketothiolase phaA) were able to accumulate poly(3HB-co-3HHx) when cultivated with even number chain fatty acids. The same trend was reported for Klebsiella aerogenes and Pseudomonas putida. 

Pathway I PhaA P-Ketothiolase Ralstonia eutropha Sudesh et al. (2000)... 

Intracellular degradation (often called mobilization) consists in enzymatic breakdown of polymers to monomers, which are then converted by o-hydroxybutyrate dehydrogenase into acetacetate. As a result of the dehydrogenase reaction, the latter is transferred to CoA, serving as a substrate for P-ketothiolase, which converts it into acetyl-CoA. Studies of intracellular degradation may be important in regard to mass production of microbial polyesters. 

Following the eatalytic reaction to form long-chain fatty acyl-CoA, the enzymatic reaction of carnitine palmitoyltranferase I (CPT-1) replaces CoA with carnitine to form fatty acylcamitine [62]. This conversion allows fatty acids to be transported from the cytoplasm to the inner mitochondrial membrane via carnitine acylcamitine translocase. Once across the inner mitochondria membrane, fatty acylcamitine is reversely converted back to long-chain fatty acyl-CoA by carnitine palmitoyltrandferase II (CPT-2) for subsequent P-oxidation [63, 64]. Each p-oxidation cycle removes a two carbon unit and involves four main enzymes 1) aeyl-CoA dehydrogenase, 2) enoyl-CoA hydratase, 3) 3-hydroxyacyl-CoA dehydrogenase, and 4) P-ketothiolase [65]. The net reaction of each P-oxidation pathway is ... 

P-Ketothiolase (encoded by phaA) condenses two molecirles of acetyl-CoA to acetoacetyl-CoA, which is then reduced to (S)-3-hydroxybutyryl-CoA by the NADPH-dependent acetoacetyl-CoA reductase (encoded by phaB). The PHB synthase (encoded by phaC in R. eutropha R.) then converts the thioester monomers into the polyoxoester PHB. The polymer aggregates to form a spherical inclusion or granule usually 50-500nm in diameter with the amorphous hydrophobic PHA polyester at the core and attached or embedded proteins at the surface, including the PHA synthase, PHA depolymerases, structural, and regulatory proteins [13,14]. 

Note CDW Cell dry weight vgb Gene encoding Vitreoscilla hemoglobin PHB synthesis genes encoding p-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase Ac Aeromonas caviae 1,4-BD 1,4-butanediol phaC c- PHA synthase gene phaC from Aeromonas caviae. 

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