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The Droppy project !

Polyhydroxyalcanoates (PHAs) are universal prokaryotic storage compounds of carbon and energy. They are accumulated into the cell as spherical inclusion bodies in case of oversupply of carbon sources. We aim to control the production and the composition of granules in Escherichia Coli. Many industrial and biomedical applications for those granules had been evocated. However, we want to focus especially on two axes :

· the granule as nano or micro-beads in order to purify a molecule of interest
· the granule as a storage system for overproduced lipids of interest such as DHA or EPA.

The great majority of microbes, simply cultivated in their usual media, are able to synthesize considerable amounts of intracellular lipids and store these molecules into large droplets easily stainable and observable. These spherical inclusions are surrounded by a mono-layer of lipids with embedded or attached proteins. To our knowledge, these lipid droplets (or "granules") had never been observed in wild-type E. coli strains. But recombinant strains have been constructed by cloning the genes responsible for the production of poly-hydroxybutyrate (PHB), an insoluble polyester, and these molecules were deposited into granules (with the diameter usually ranging between 100 and 500 nm and 5-10 granules per cell). These recombinant bacteria are able to accumulate as much as 80% of their dry weight in PHB.

Considering this very important result, the working hypothesis at the origin of our project is that E. coli (and maybe any cell) is able to produce granules when large amounts of hydrophobic molecules are synthesized.

In a first step, we will test this hypothesis by constructing E. coli strains which produce other hydrophobic molecules and examining granule synthesis. For this, we intend to construct a new part : a strong promoter sensitive to the shaking speed of the water bath in which the bacteria are cultured. Our objective is to trigger lipid synthesis and granule production just by varying this shaking speed.

The second step of our project is the use of the PHB granules as a tool to produce and purify recombinant proteins. Various proteins, such as phasins, are attached to the external surface of the granules. Our objective is to construct a part able to target proteins of interest to this surface. Moreover, in order to facilitate purification, the sequence of a self-cleaving protein (intein) will be added to this part.

From "Bacterial polyhydroxyalkanoate granules: biogenesis, structure, and potential use as nano-/micro-beads in biotechnological and biomedical applications." Grage K., Jahns A. C., Parlane N., Palanisamy R., Rasiah I. A., Atwood J. A. and Rehm B. H. A. 2009. Biomacromolecules 10, 660-669.

In addition, we intend to develop a modelisation project. In plants and bacteria, the reactions involved in fatty acid synthesis are catalyzed by separate monofunctional enzymes. But in animals, evolution has done synthetic biology and the different enzymes are integrated into a single multifunctional polypeptide in which substrates are handed from one functional domain to the next. We have begun to analyse the work of evolution in animals in order to design multifunctional bacterial enzymes that integrate discrete monofunctional enzymes. We hope that these enzymes could promote the synthesis of particular lipids, such as tri-acyl glycerols, and store these molecules into granules.