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Team:INSA-Lyon/Project
From 2010.igem.org
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 potential applications (see Uses):
· 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.
We aim to regulate the granule production in E. coli thanks to our favorite new part: a strong promoter sensitive to the shaking speed and the temperature of the water bath. By controlling this production, our team focuses on two final purposes: (1) the granule as a storage system for overproduced lipids with medical applications, such as DHA or EPA and (2) the granule as self-cleaving micro-beads in order to purify a recombinant protein of interest. In bacteria, three separate monofunctional enzymes are required for PHA synthesis. In order to improve this pathway, we intend to model a single multifunctional enzyme based on the study of natural evolution of fatty acid synthesis in animals.
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.
