The nature of PHA inclusions
PHAs are synthesized by various microorganisms as carbon and energy storage compounds, which consist of (R)-3HA monomer unit. The 3-hydroxyalkanoic acids are all in the R configuration due to the stereospecificity of the polymerizing enzyme, PHA synthase. The most well known member of PHA is poly(3-hydroxybutyrate) [P(3HB)], containing repeat units of (R)-3HB. The monomers are polymerized into high molecular weight polymers in the range of 200,000 to 3,000,000 Dalton, depend on the microorganism and growth conditions.
Biosynthesis of poly(3-hyroxybutyrate-co-3-hydroxyvalerate)
[P(3HB-co-3HV)] from palm oil and 3HV precursors using
Cupriavidus necator H16
PHA exists as discrete inclusions in the cell cytoplasm and may be visualized quite clearly under phase contrast light microscope due to their high refractivity . When thin sections of PHA-containing bacteria are observed by transmission electron microscopy, the PHA inclusions appear as electron-dense bodies. Native PHA inclusions can be stained with Sudan black B or Nile Blue A while Nile Red can be used to detect PHAs directly in growing bacterial colonies. Chemical analysis such as gas chromatography (GC) or nuclear magnetic resonance spectroscopy (NMR) analysis is often required to determine its monomer composition.
Types of PHA
In the biosynthesis of PHA, the polymerizing enzyme, PHA synthase (PhaC), has been identified as the key enzyme which determines the type of PHA synthesized by a microorganism. Three different types of PHA synthases can be distinguished with respect to their substrate specificities and primary structures.
The first type of PHA synthases, represented by the well characterized PHA synthase of R. eutropha, includes those that are active towards short-chain-length (SCL) HA monomers. The SCL HA monomers are defined as the (R)-enantiomers of 3-, 4-, and 5-HA containing 3 to 5 carbon atoms.
The second type of PHA synthases has been characterized as those that efficiently incorporate larger (R)-3HA monomers containing 6 to 14 carbon atoms (termed medium-chain-length [MCL] HA). This type is represented by the two PHA synthases of P. oleovorans.
The third type of PHA synthases has been characterized based on the fact that, unlike the first and second types which consist of only one subunit of about 60 to 70 kDa, the third type consists of two subunits designated as the C-subunit (~40 kDa) and E-subunit (~40 kDa) . The substrate specificities of PHA synthases in this group are apparently not very well defined, but generally they seem to prefer SCL HA .