3-Ketothiolase phaA

R. eutropha, formerly known as Alcaligenes eutrophus, has been used for the commercial production of P(3HB-co-3HV) [72]. This bacterium grows well in a relatively inexpensive minimal medium and accumulates a large amount of P(3HB) under the unbalanced growth condition. In R. eutropha, acetyl-CoA is converted to P(3HB) by three enzymes (genes) /J-ketothiolase (phaA), aceto-acetyl-CoA reductase (phaB), and PHA synthase (phaC) [6]. 

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. 

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. 

Fig. 2.3 Biosynthesis pathway of A P(3HB) B P(3HB-co-3HV) C P(3HB-co-3HHx) via fatty acid /S-oxidation and D P(3HB-co-3HHx) via fatty acid de novo synthesis. PhaA, f -ketothiolase PhaB, NADPH dependent acetoacetyl-CoA reductase PhaC, PHA synthase PhaG, 3-hydroxyl-ACP-CoA transferase PhaJ, (J )-enoyl-CoA hydratase FabG, 3-ketoacyl-CoA reductase (Sudesh et al. 2000)...

So far, biosynthesis of PHA can be summarized in eight pathways (Fig. 4, Table 1). The first pathway involves the three key enzymes (3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHA synthase encoded by genes phaA, phaB, and phaC, respectively. Ralstonia eutropha is the representative of this pathway. An associated pathway involving PHA degradation catalyzed by PHA depolymerase, dimer hydrolase, 3-hydroxybutyrate dehydrogenase, and acetoacetyl-CoA synthase helps regulate PHA synthesis and degradation. The associated pathway was found in strains of Aeromonas hydrophila. Pseudomonas stutzeri, R. eutropha, and Pseudomonas oleovorans (Sudesh et al. 2000). 

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

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]. 

P(3HB) is one of the most commonly produced and most extensively studied SCL-PHA. Fig. 8.3 elucidates the enzymatic pathway for the biosynthesis of P(3HB). The biosynthetic pathway of P(3HB) synthesis involves three major reactions and three key enzymes. The first reaction is the condensation of two acetyl-CoA molecules (derived fiom the TCA cycle) to form acetoacetyl CoA. This reaction is catalysed by the enzyme fi-ketothiolase, which is encoded by the phaA gene. The second reaction is the reduction of acetoacyl CoA to (R)-3-hydroxybutyryl CoA. This reaction is catalysed by the NADH-dependent enzyme acetoacetyl-CoA... 

Figure 7.7 Common metabolic pathways that are involved in the biosynthesis of PHA in microorganisms. FabC malonyl-CoA acyl carrier protein (ACP) transcylase, FabD malonyl-CoA-ACP transacylase, FabG 3-ketoacyl-CoA reductase, PhaA P-ketothiolase, PhaB NAOH-dependent acetoacetyl-CoA reductase, PhaC polyhydroxyalkanoates synthase, PhaG 3-hydroxyacyl-ACP GoA transferase, PhaJ (R)-enoyl-GoA hydratase and TCA tricarboxylic acid...

Figure 1. SCL-PHA production from non-related carbon sources. A. The tricarboxylic acid cycle. B. Poly-3-hydroxybutyrate [P(3HB)] monomer supply via the fatty acid biosynthesis enzymes, FabD, FabH, and FabG. C. P(3HB) monomer supply mediated by the beta-ketothiolase enzymes, PhaA or BktB, and the NADPH-reductase, PhaB. D. Poly-3-hydroxyvalerate (P3HV) monomer supply mediated by threonine deaminase (IlvA), pyruvate dehydrogenase, BktB, and PhaB. E. Poly-4-hydroxybutyrate monomer supply mediated by succinate dehydrogenase (SucD), 4-hydroxybutyrate dehydrogenase (4HbD), and acetyl transferase (Catl or Cat2).

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