Biochemical pathways involved in the metabolism of PHA
In order to tap the full potential of microbial systems for PHA production, it is necessary that the existing metabolic pathways in a particular microorganism are modified. This is to ensure that the major portions of the supplied carbon sources are channelled towards PHA biosynthesis. New pathways can be constructed by introducing relevant genes into suitable microorganisms. Likewise, unnecessary pathways can be shutdown by inactivating the enzyme(s) involved in a certain reaction. Such manipulations have to be carried out judiciously to achieve maximum PHA production in the shortest possible time using cheap and readily available carbon sources, without compromising the cell growth.
Acetyl-CoA as precursor
Along with the type of carbon source and the specificity of the PHA synthase, the metabolic pathways play a crucial role in determining the type of PHA that can be produced by a particular microorganism. Most of the P[3HB] producing microorganisms possess pathway which acetyl-CoA is converted into (R)-3-hydroxybutyryl-CoA and subsequently polymerized by the PHA synthase. In some microorganisms, (S)-isomers of 3-hydroxybutyryl-CoA are generated instead of the (R)-isomers. Since the PHA synthase is active only towards the (R)-isomers, additional reaction steps catalysed by enoyl-CoA hydratases are present in microorganisms such as Rhodospirillum rubrum to convert the (S)-isomers into the (R)-isomers .
Intermediates in Fatty acid ß-oxidation pathway
Fatty acid ß-oxidation pathway is known to generate substrates that can be polymerised by the PHA synthases of pseudomonads. Pseudomonads that belong to the rRNA-homology group I can synthesise PHAMCL from various alkanes, alkanols, and alkanoates. The monomer composition of the PHAMCL produced is often related to the type of carbon sources. Most of the pseudomonads belonging to this group, except P. oleovorans, can also derive (R)-3-hydroxyacyl-CoA substrates for PHA biosynthesis from unrelated carbon sources such as carbohydrates.
Intermediates in Fatty acid de novo biosynthesis pathway
Among the various metabolic pathways that are involved in PHA biosynthesis, the fatty acid de novo biosynthesis pathway is of particular interest because of its ability to supply various types of hydroxyalkanoate monomers from simple carbon sources such as gluconate, fructose, acetate, glycerol and lactate. It can be envisaged that the potential future production of PHAMCL by using photosynthetic organisms will benefit through the exploitation of such pathways. This is because acetyl-CoA is the starting material (in fatty acid de novo biosynthesis pathway) that is used to generate hydroxyalkanoate monomers for PHAMCL biosynthesis, and acetyl-CoA is a universal metabolite present in all living organisms. However it must be noted that the intermediates of fatty acid de novo biosynthesis pathway are in the form of (R)-3-hydroxyacyl-ACP, which is not recognised by the PHA synthase.
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 tricarboxylic acid (TCA) cycle . By providing external precursor substrates such as 4-hydroxybutyric acid, 1, 4-butanediol, and y-butyrolactone to certain wild type [39, 40, 41, 42] and recombinant microorganisms [43, 44], 4HB monomers can be incorporated more efficiently.