A deficit in frataxin (FXN) leads to Friedreich's ataxia (AF), the most common hereditary ataxia. The primary consequence of FXN deficiency is a decrease in the biogenesis of Fe-S centers. Biogenesis of Fe-S centers is an essential process present in all living organisms required for the functioning of a myriad of proteins involved in key cellular processes. In humans the biogenesis of Fe-S centers is provided by the ISC machinery, a multiprotein machine located in the mitochondria. ISC machinery is also present in prokaryotic organisms such as E. coli. Prokaryotic and eukaryotic ISC proteins are highly homologous in their primary sequence, and their molecular mechanism has been found to be similar. Formation of the Fe-S center is catalyzed by the assembly complex which contains a sulfur-producing cysteine desulfurase, a scaffold protein on which the Fe-S center assembles and FXN plays a regulatory role. Up to now, there is no satisfactory treatment to restore the biogenesis of Fe-S centers in patients with AF. This project aims to fill this gap by finding ways to restore the biogenesis of Fe-S centers in FXN-deficient cells. Our working hypothesis - based on previous results, including ours and those of other laboratories - is that modifying the components of the human ISC assembly complex such that it can function without FXN is possible. Recruiting existing components to endow them new properties or assembling them in non-natural systems are the principles of the engineering sciences exploited by "synthetic biology". The objective of the FRACOL project is to apply such principles by exploiting biodiversity, genetic tools and our mastery of the physiology of E. coli to produce "non-natural", heterologous and/or evolved ISC machines that can operate without FXN. Our research will develop along two main axes. The first axis, essentially genetic, will aim to produce "non-natural" ISC machineries. The first strategy involves using E. coli as a chassis to construct hybrid ISC machineries made up of E. coli ISC components operating with "heterologous" ISC components identified in biodiversity by phylogenomy and genomic analysis. Heterologous ISC components will be sought in organisms without FXN detectable by genomic analysis. Another strategy will be to evolve the ISC machinery of E. coli in such a way that it can operate without FXN. Mutagenesis strategies coupled with various selecting / screening protocols will be used. Finally, a third strategy will be to construct an E. coli strain using human ISC machinery to function. Such an “humanised” E. coli will then be submitted to genetic selection protocols to modify the human ISC machinery operating in E. coli. The second axis will essentially be an approach exploiting the screening of chemical compounds. We will screen, from chemical libraries, the molecules allowing the ISC machinery to function independently of the FXN. The identified molecules will be validated in FXN-deficient cell models, and mice models of Friedreich's ataxia. Biochemistry and biophysics studies will then characterize the new machineries or the effect of chemical compounds in order to understand how the need for FXN was by-passed, and rules will eventually be extracted as how to modify ISC system for them to function without FXN. The project brings together three internationally recognized teams with remarkable complementarity. For the first time, the power of bacterial genetics will be used to shed light on how human ISC machinery could assemble Fe-S centers in the absence of FXN. Moreover, by combining biochemical and biophysical methods, we will establish the molecular bases of the mechanisms allowing to by-pass FXN.