How to use the granule ?
Composition of the granules
Figure 1 :PHB granule. (Grage et al., Biomacromolecules, 2009)
The formation steps of those granules are not well determined, but their final structure and composition have been described. The three PHB biosynthesis genes are organized in one operon in the Ralstonia eutropha H16 strain: the phaCAB operon. In addition to the three essential enzymes for the PHB granules biosynthesis, other proteins contribute to the regulation and the formation of the granules. They can be separated in three classes: the depolymerases (PhaZ), the regulatory proteins (PhaR) and the phasins (PhaP). They are located on the fatty surface of the granule (see on Figure 1).
PhaPs, also called phasins, are proteins regulating the size and the morphology of granules and preventing the fusion of PHB granules by formation of a protein layer between the hydrophobic polymer and the hydrophilic cytoplasm. Their expression is regulated by the PHB accumulation, thanks to a repressor, PhaR. Studies show that PhaR can be bound by growing PHB granules during PHB accumulation. A low concentration of soluble PhaR allows the expression of more PhaP and PhaR. Once all binding sites for PhaR on the PHB granules are occupied by PhaR and PhaP, excess soluble PhaR binds to DNA upstream regions of PhaP and PhaR, repressing the expression of the two proteins. 5% of the granule is composed by these proteins, called Granules Associated Proteins.
Thanks to the understanding of the PHA granule synthesis, we aim on the first time to synthesize granules of other lipids according to the theory that any hydrophobic lipids would accumulate in granules under the control of chosen enzymes. We want to use E.coli as a factory of lipids with a medical interest such as EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid). The extraction of those lipids would be simplified by the accumulation in granules.
The production of EPA, which is one of the principal omega-3 fatty acids, represents a real interest because the body is not able to synthesize itself this fatty acid. We can find them in our alimentation with fish or oil, but our consumption is often not sufficient to fulfill our needs. The body can metabolize EPA from ALA alpha-linoleic acid but it is a limited pathway and EPA is also a precursor of DHA, an other important omega-3 fatty acids. Some studies showed the benefits of the omega-3-fatty acid like EPA or DHA on health: they reduce for example the risks of cardio-vascular diseases and neuro-degenerative diseases such as Parkinson and Alzheimer diseases. That’s why it’s promising for medicine to find another way to produce them.
Concerning the massive production of this fatty acid, some researches showed that microalgae are able to synthesized EPA and DHA. However, because of their photo-autotroph metabolism, the production of biomass microalgae large-scale requires the conception of specific bioreactors, photo-bioreactors, which represents even today a technological challenge. Thus it is necessary to confirm the economic viability of the production industrial processes of EPA and DHA by microalgae. The production of fatty acid by bacteria like E.coli could be an important innovation since E.coli is a very well known production system and usually used to overproduce molecules of interest.
Figure 2 : PHB-intein method of affinity-based protein purification
(Banki et al., Protein Science, 2005)
The synthesis of PHB itself is interesting because it can be used in many fields. But, we focused more especially on the structure of the granule which can be seen as micro-beads purification way thanks to the proteins phasins on its surface. It has been shown that this protein has the ability to be inserted into the granule membrane when it is in the cytoplasm. (Banki et al., 2005). The fusion between the phasin and a molecule of interest would allow the binding of this construction on the surface of the granule. The extraction of the granule also means the extraction of the molecule of interest. To facilitate the purification process, we added a self cleaving sequence between the phasin and the molecule of interest (see below on Figure 2).
PHB-intein method of affinity-based protein purification: cells containing two plasmids, one for biosynthesis of PHB granules and another for expression of the phasin-intein tagged product protein, are grown to produce PHB and express the affinity fusion. Harvested cells are lysed and centrifuged to separate soluble components (1B). The insoluble PHB granules with the PHB-bound fusion protein are washed and resuspended in a cleavage-inducing buffer for release of the product protein (2B). A final centrifugation separates the PHB granules and associated proteins from the cleaved product protein, leaving only the product protein in the soluble fraction (3B).
Go to the page Protocols to have more informations about the extraction of the granule and the intein cleavage.