Carbon Sources for the Production of Polyhydroxyalkanoates

On the other hand, the two-step process involves the direct conversion of plant sugars and plant oils into polymers hy microorganisms. At present, the biosynthesis of PHA is largely carried out through this two-step process. Compared with the three-step process of polymer production, the two-step process can be more cost-effective 

Although it was known that the intermediates of the yS-oxidation cycle are chaimelled towards PHA biosynthesis, only recently the precursor sources were identified. In A. caviae, the y3-oxidation intermediate, trans-2-tnoy -CoA is converted to (R)-3-hydroxyacyl-CoA via (R)-specific hydration catalysed by an (R)-specific enoyl-CoA hydratase [125, 126]. Subsequently, Tsuge and co-workers [127] reported the identification of similar enoyl-CoA hydratases in Pseudomonas aeruginosa. In the latter case, two different enoyl-CoA hydratases with different substrate specificities channelled both SCL and MCL enoyl-CoA towards PHA biosynthesis. In recombinant . coli it was further shown that 3-ketoacyl-CoA intermediates in the )8-oxidation cycle can also be channelled towards PHA biosynthesis by a nicotinamide adenine dinucleotide phosphate dependent (NADPH-dependent) 3-ketoacyl-ACP reductase [128]. A similar pathway was also identified in P. aeruginosa [129]. In addition, it was also reported that the acetoacetyl-CoA reductase (PhaB) of R. eutropha can also carry out the conversion of 3-ketoacyl-CoA intermediates in Pathway II to the corresponding (R)-3-hydroxyacyl- CoA in E. coli [130]. The results clearly indicate that several channelling pathways are available to supply substrates from the y3-oxidation cycle to the PHA synthase. This explains why it was not possible to obtain mutants that completely lack PHA accumulation ability, unless the mutation occurred in the PHA synthase gene [131]. 

Besides the three main pathways mentioned above, there are several other metabolic pathways that can be manipulated to produce substrates for PHA biosynthesis. In recombinant E. coli, it has been shown that 4-hydroxybutyryl-CoA can be derived from the intermediates of the tricarboxylic acid (TCA) cycle [137]. By providing external precursor substrates such as 4-hydroxybutyric acid, 1,4-butanediol and y-butyrolactone to certain wild type [59, 60, 62, 63] and recombinant microorganisms [138], 4HB monomers can be incorporated more efficiently. 

In order to elucidate the mechanism of PHA synthase, various site-specific [141,142] and random mutagenesis [143] studies have been carried out. Results show that many of the amino acid substitutions that affected PHA accumulation occurred in the conserved regions within an oc/p hydrolase fold , while changes in the sequence of the first 100 amino acids at the N-terminal region did not show any significant effect. The ct/p hydrolase fold is characteristic of a protein superfamily that includes lipases [144]. 

Initially it was thought that the PHA synthase catalytic mechanism might resemble that of fatty acid synthases [145,146]. Present knowledge dictates that a lipase-based catalytic mechanism is perhaps more suitable as a model of PHA synthase [147]. 

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