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Research Topic, LBC

Biotechnological applications

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Published on 29 July 2016
Daniel GARCIA & David PIGNOL

Researchers : Monique SABATY, Nicolas GINET, Arjan DeGROOT Pascal ARNOUX​

Technician/Engineer : Camille Escoffier, Sandrine GROSSE, Géraldine ADRYANCZYK, Catherine BRUTESCO  & Sandra PREVERAL
​Our fundamental research carried out on different bacterial models are feeding a set of applied projects that are developped in the context of different partnership-contracts on environmental biotechnology and technology for health (1-3).

Biodetection

In the course of the COMBITOX project, we focused on conceiving an in-line multiparametric prototype for the surveillance of water networks using biosensors. Thanks to our expertise in the field of bacterial responses to metal, we developed bioluminescent whole-cell biosensors based on reporter gene for the detection of toxic metals (cadmium, mercury, arsenic, nickel, cobalt, etc), with genetic improvement of the dose-responses and the specificity. We also addressed biosensor conservation over a period of time compatible with the autonomy of the device requested by the end-user, and developed alternative near-infrared fluorescent reporter candidate to perform biodetection in turbid and complex water samples. Moreover, we characterized the use of Deinococcus deserti as cellular chassis to offer desiccation as a cost-effective solution to biosensors long-term storage and to open the field to metallic radioisotopes detection. Our resulting prototype allows the detection of bioavailable toxic compounds impacting human health through the drinking water network. 

Bioremediation 

Several strains studied in the lab have been shown to accumulate or degradate various pollutants (including heavy metal ions and organophosphorus pesticides) (4-6). In this context, we first took advantage of the discovery of a new bacterial metallophore and our expertise in magnetotactic bacteria to develop an original bioremediation tools for cobalt bioaccumulation (Project BAMACO, Toxnuc). Heterologous expression of genes encoding the enzymatic machinery responsible for staphylopine synthesis increases both the bacterial resistance towards cobalt and the intracellular sequestration of the metal in two strains of magnetotactic bacteria. These engineered strains represent a highly efficient bio-accumulation system that can be easily recovered from the medium by using a magnet.​

Second, thanks to our knowledge accumulated on photosynthetic bacteria, we have been coordinating a collaborative project funded by the Life+ program (an EU's financial instrument) to demonstrate an innovative process for treating phytopharmaceutical effluents (pesticides) with the development of demonstrators on site. The treatment is based on the use of photosynthetic microorganisms shown in the lab to be efficient to degrade active molecules, including organophosphates. The process consists in a series of three lagooning basins supplied via a washing station (for farm machineries) that recovers phytopharmaceutical residues. The basins are unventilated and cascading, and are seeded with a bacterial consortium selected by the laboratory to degrade active molecules in the effluents. Monitoring of the pesticides charge conducted in different farms indicates an almost complete degradation of most of the active molecules discharged into the ponds. Conservation in the most suitable packaging for a simple and easy manipulation by non-specialist users is also addressed and the creation of a startup company to exploit this process is currently under consideration.​

For more updated information on this project, connect to the following link: http://www.lifephytobarre.eu/​

Green Chemistry

We also investigate the magnetization properties of Magnetotactic bacteria for the construction of recyclable biocatalysts. Magnetosomes functionalization can thus be achieved by translational fusion coupling a membrane anchor to an enzyme of interest (7). As a result, biocatalysts are synthesized and immobilized on the magnetic nanoparticles by the cellular machinery, and they can be simply recovered  after purification of the magnetosome using magnetization,. The applications of such a process are numerous both in environmental and health biotechnologies.

Biotechnology for health

Magnetosomes are unique biogenic nanoparticles with a large potential in health biotechnology. Isolated magnetosome crystals are indeed superior to synthetic particles because of their unique characteristics: (i) a perfectly crystalline nanocrystal of magnetite, with contrasting properties for magnetic resonance imaging (MRI); (ii) magnetic properties that allow easy separation or guidance; (iii) a natural lipid bilayer coating the nanoparticles, ensuring their solubilization; (iv) a possible functionalization of the lipid surface with biological functions for cellular targeting or in situ enzymatic catalysis. In the last period, we coordinated a collaborative project (MEFISTO, ANR P2N) that focused on the use of magnetosomes in the localized treatment by hyperthermia of cerebral tumors assisted by high-field MRI diagnostics (in collaboration with Neurospin, CEA Saclay). While MTB are difficult to grow in large quantities, we first succeeded in setting up the semi-automated, large scale production in 7 liters bioreactors of two magnetotactic strains, Magnetospirillum magneticum AMB-1 and Magnetovibrio blakemorei MV-1. We also devised new extraction, purification and characterization protocols to obtain large quantities of magnetosomes required for further biotechnological developments. We demonstrated that the systemic injection of purified wild-type magnetosomes reveal the mouse brain vasculature (ACL7.53). Based on previous magnetosomes functionalization experiments for bioremediation (ACL7.5), we genetically functionalized the outer surface of the magnetosomes membrane with a RGD peptide known to specifically target anb3 integrin receptors generally expressed at the surface of cancer cells during angiogenesis. The ability of purified RGD-magnetosomes to target these receptors was evidenced in vitro on U87 cells (human glioblastoma) overexpressing the anb3 integrin receptors by immuno-, histo- chemical staining and fluorescence microscopy while in vivo MRI at 11.2 T revealed the enhanced retention time of the RGD-magnetosomes within the tumor compared to the wild-type magnetosomes (publication under submission). These results pave the way to future treatment of glioblastoma-bearing mice with magnetic hyperthermia after RGD-magnetosomes systemic injection and under MRI diagnostics.




PHYTOBARRE demonstrator consisting un a washing area coupled to a series of three lagooning basins seeded with photosynthetic bacteria selected for pesticide treatment

 

functionnalisation of the magnetosome
Crédit : Nicolas Ginet/CEA



In vivo MRI experiments on a mouse brain acquired before and after tail vein injection of magnetosomes​
Crédit : Nicolas Ginet/CEA

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Collaborators
  •  S. Rebuffat (Museum)
  • S. Mériaux (LRMN, Neurospin)
  • Société AP2, Société Nanobactérie
  • Société Barre.
  • Marie Carriere, CEA DRF Grenoble
  • G. Lambert (LESA, Aix-en-Provence) ​

Fundings 
  • ​ANR COMBITOX (Ecotech, 2011-2015), coordinateur D. Pignol
  • ANR MEFISTO (P2N, 2013-2016), coordinateur D. Pignol
  • LIFE- PHYTOBARRE (Life+, 2013-2016), coordinateur D. Garcia 
 
Pu​blications

1- Garcia D & Pignol D (2012) Biofutur. Un éclairage bactérien sur la détection des toxiques.
2- Garcia D & Pignol D (2012) Biofutur. Des micro-organismes domestiqués pour dépolluer.
3- Garcia D & Pignol D (2012) Biofutur. Exemple d’un procédé biotechnologique de traitement des effluents phytosanitaires.
4- François F, Lombard C, Guigner JM, Soreau P, Brian-Jaisson F, Garcia D, Molinier AL, Pignol D, Peduzzi J, Zirah S, Rebuffat S. (2012) Bacteria with toxic metal biosorption capacities for bioremediation applications. A.E.M. 78(4):1097-106.
5- Berne C, Pignol D, Lavergne J, Garcia D. (2007) Cytochrome P450 CYP201A2 from Rhodopseudomonas palustris plays a key role in the tributyl phosphate biodegradation. Appl. Microbiol. Biotechnol 77(1):135-44
6- Dreyfus C, Cavelier F, Martinez J, Laoure M, Arnoux P, Pignol D. Métallophore dérivé de la nicotianamine et ses procédés de fabrication. brevet FR0901574
7- Ginet N, Pardoux R, Adryanczyk G, Garcia D, Brutesco C, Pignol D. (2011) Single-step production of a recyclable nanobiocatalyst for organophosphate pesticides biodegradation using functionalized bacterial magnetosomes. PLoS One 6(6):e21442
8- Mériaux S., Boucher M., Marty B., Lalatonne Y., Préveral S., Motte L., Lefèvre C.T., Geffroy F., Lethimonnier F., Péan M., Garcia D., Adryanczyk G., Pignol D., Ginet N. (2015) « Towards brain molecular imaging with bacterial magnetosomes and 17.2 T MRI scanner» Adv Healthc Mater. Feb 13. doi: 10.1002/adhm.201400756.