**2.3 Biosynthesis of PHA**

PHAs are produced from two molecules of acetyl-CoA by three enzymatic reactions. The classical polyhydroxybutyrate (PHB) biosynthesis pathway consists of the following reactions:


However, apart from the classical pathway, there are other biosynthetic pathways involved in PHA production that differs based on the substrates, enzymes, and microorganisms used. The enzyme PHA synthase plays the most crucial role in PHA synthesis since it can polymerize 3-HA units obtained from different pathways such as fatty acid β-oxidation pathway, methylmalonyl-CoA pathway, and de novo fatty acid synthetic pathway [21, 22]. Numerous studies conducted on heterophilic bacteria revealed the classification of PHA synthase based on the specificity of 3-HA (C-Chain Length) substrate, amino acid sequence, and constituent subunits to have four classes [23]. Class I PHA synthases are encoded by phaC and polymerize scl-3HA units, monomers with approx 64 kDa MW. Class II PHA synthases polymerize mcl-3HA and are also encoded by phaC genes. These are monomers and have similar MW of 63 kDa. Class III PHA synthases are heteromeric with 40 kDa two subunits encoded by phaC and phaE genes each. They polymerize scl-3HA units. Class IV PHA synthases are similar to Class III and are encoded by either phaEC genes or phaRC genes. They polymerize scl-3HA to mcl-3HA and scl-3HA alone, respectively.

The acetyl-CoA utilized in the classical pathway of PHB synthesis is acquired as precursors derived from the tricarboxylic acid (TCA) cycle. This type of pathway is most commonly found in cyanobacteria, archaea, and heterophilic bacteria such as *Cupriavidus metallidurans*. Lipid metabolism is also used for the production of PHA which are mostly medium chain length (MCL) —PHAs. Different hydroxyalkanoates are generated from the β-oxidation pathway of fatty acids by the biotransformation of alkanes, alkenes, and alkanoates. The conversion of the β-oxidation intermediate trans-2-enoyl-CoA into (R)-hydroxyacyl-CoA is catalyzed by an R-specific enoyl-CoA hydratase (encoded by phaJ gene) and is the crucial step in this type of pathway. Studies conducted on *Aeromonas caviae* and *Pseudomonas putida* strains reported the (R)-specific manner of action of the phaJ enzyme [24, 25]. The PHA synthase (encoded by phaC genes) polymerizes (R)-hydroxyacyl-CoA into PHAs. MCL -3HA is produced in this type of pathway where both sugars and lipids are utilized. Glycolic precursor and fatty acid biosynthesis intermediates are converted to 3-hydroxyacyl-ACP by 3-hydroxyacyl-ACP-CoA transferase and malonyl-Coa-ACP transacylase. These key enzymes are encoded by phaG gene and are (R)-specific reactions by acyl-ACP-CoA transacylase. The 3-hydroxyacyl-ACP is converted into 3-hydroxyacyl-CoA and then polymerized to PHAs by PHA synthase.

Apart from the biosynthesis pathways and carbon source, other nutrients such as phosphate, nitrogen, oxygen, and sulfur also play a major role in PHA accumulation [26]. Limiting nitrogen and/or phosphorus with an excess carbon source is favorable for cell growth, along with C: N ratio changes showing better beneficial stress for PHA accumulation [15, 27, 28]. Under nitrogen deprivation, the conversion of αketoglutarate to glutamate is decreased causing accumulation of NAD(P)H by absorption of ammonium ions into cells. Similarly, the supplement of citrate reduces citrate synthase activity, thereby increasing the concentration of NAD(P)H. These high concentrations of NADPH result in increased PHB production since the reduction of acetoacetyl-CoA to R-3-hydroxybutyryl-CoA is increased [29]. Limiting phosphorus to a minimum level needed for cell maintenance restricts the Krebs cycle by promoting NADH accumulation, inhibiting citrate synthase and isocitrate dehydrogenase with increased acetyl-CoA. Nutritional stress induced by phosphorus limitation is sometimes more significant than nitrogen as a limiting factor in cyanobacteria and proved to be a good strategy for inducing PHA production [29] (**Figure 2**).
