Natural products are important providers of drug leads in the occidental society and have played key roles in biological sciences and medicine due to their privileged ability to interact with life components. However, it is obvious that they cannot be sustainably purified from harvested living organisms, on the large industrial scale, for socio-economical purposes. Access to pharmaceutically relevant natural compounds or improved analogues can thus mainly be obtained by chemical (synthetic chemistry) and/or biological (synthetic biology) means. Meanwhile, the increased incidence, the recrudescence and the emergence of old or new diseases worldwide (especially in Europe or the entire occidental society) – like cancer, viral, microbial multi-drug resistant infections, age-related neurological disorders or diabetes – impose new efficient strategies to discover and develop new drug leads, and thus impose new productive challenges to synthetic chemists and biologists. Even old drug repositioning which is today systematically considered by pharmaceutical companies may be limited, while personalized medicines and biological therapies are in no way generalizable, rendering the search of new molecules with unmatched chemical space (which is a characteristic of natural products) more than ever needed. During this ANR MRSEI project, ten academic partners from four European countries and additional non-academic actors will be invited to join an educational research proposal centered on medicinal natural product synthetic chemistry and biology, to apply the European MSCA-ITN-2017-ETN call. The Synspired ANR program will be co-funded by the GDRI i-NPChem launched on January 2016 to provide the best conditions needed for a successful tentative. The ANR financial support will be used to organize scientific and management meetings aiming at the preparation of our European project, as well as specific missions for French partners in the same way, and the organization of a Summer School in 2017 to anticipate or kick off the European ITN project.

Multiple bacterial natural products including the pepteridine virulence factors and the therapeutically-relevant antibiotic virginiamycin M, are biosynthesized at the intersection between primary and specialized metabolism. In these cases, primary metabolic α-ketoacid dehydrogenase complexes (KADHs) provide essential acyl building blocks to multienzyme complexes of specialized metabolism, including modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). More remarkably, in certain pathways, the KADH components are fully integrated into the PKS/NRPS megaenzymes. At present, nothing is known about the sequence, mechanistic and architectural adaptations that were required relative to the ancestral KADHs to afford such chimaeras – information which is necessary for creating novel types of hybrids in the laboratory by genetic engineering. In this context, the present German-French collaborative project aims to investigate this type of system in detail. Specifically, we will: (i) generate an exhaustive catalog of specialized metabolic pathways that incorporate KADH machinery using genome mining; (ii) structurally characterize the products of newly-identified systems by heterologous expression; (iii) use ancestral protein reconstruction to propose a reasonable evolutionary trajectory to present day KADH enzymes; (iv) deploy an integrative structural biology approach to elucidate key architectural features of multienzyme-integrated KADHs (i.e. oligomerization state, stoichiometry of KADH component binding, and interactions with partner domains within the multienzymes); and (v) exploit the obtained fundamental insights to genetically engineer biosynthetic systems, towards the goal of generating bespoke natural product analogs bearing KADH-derived moieties. The proposed project follows on from previous successful collaboration between three of the partner laboratories, and is fully anchored in all groups’ strong, highly-complementary expertise.

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products mainly produced by bacteria. These peptides contain myriad post-translational modifications which confer them with a wide structural diversity and major biological properties (e.g. antibiotic and anti-cancer activities). Among the diverse enzymes that install post-translational modifications in RiPPs, radical SAM enzymes have recently emerged as key biocatalysts. These enzymes, which form one of the largest superfamily of enzymes, catalyze a broad range of chemically complex and often unprecedented biochemical transformations. In the frame of the SAM4RiPP project, we aim to explore the mechanism and biosynthetic potential of novel radical SAM enzymes in order (i) to better understand how these fascinating biocatalysts interact and modify their peptide substrates and (ii) to develop novel hybrid RiPPs with engineered properties. Indeed, thanks to their biosynthetic logic, RiPPs are very attractive products to develop, based on synthetic biology approaches, innovative antibiotics and to tune the human microbiota.

Cyanobacteria are present in a large range of habitats and climates, including extreme conditions going from deserts to arctic lakes. They are equipped with all the physiological mechanisms needed to survive to extreme and fluctuating environments allowing them to be the predominant species in ecosystems under specific environmental conditions. Under certain conditions, a sudden and rapid growth of one or some cyanobacterial species is induced leading to blooms. There is a direct relationship between the frequency of cyanobacterial blooms and the augmentation of available nutritive resources caused by human activities. Climate changes also favor the appearance of cyanobacteria blooms suggesting that this phenomenon will be amplified in the future. The cyanobacterial blooms disrupt the functioning of the continental aquatic ecosystems with serious consequences for the production of drinking water, or recreational aquatic activities. These disturbances are largely connected to the capacity of some cyanobacteria to synthesize toxins hazardous for human and animal health. Among them, the microcystins (MCs) are the most common as they are produced by the most common bloom-forming species Microcystis and Planktothrix (cHAB: cyanobacterial Harmful Algal Bloom). The concentration of toxins present in each bloom varies. Indeed, for a toxic species, only some strains present in the bloom synthesize toxins, and in addition, in these toxic cells, toxin biosynthesis depends on environmental conditions. Thus, despite that the survey of cyanobacterial cells and toxins is one request of safety agencies in Europe and France, it is impossible to predict the concentration of toxins only from biomass data. This impossibility of prediction represents big costs for the water end-users and a health risk for populations. For this reason, it becomes urgent to understand the determinism of the toxin production during the blooms and one way to achieve this goal is to found what are the factors regulating toxin synthesis and to elucidate the functional role of toxins. Several stress conditions increases cyanotoxin synthesis but it is not clear if there is a direct effect of stress factors or an indirect effect via photosynthesis and/or photoprotection. It is also not clear, why cyanotoxin producers are more resistant to stress. Is it a direct effect of the toxin or a photoprotective mechanism up-regulated by their presence? Thus, it is crucial for the understanding of cHAB to examine the fluctuation of toxic cyanobacterial sub-populations and toxin synthesis in relationship with the rates of photosynthesis and protection mechanisms in strains isolated from natural environment and usually present in blooms. This is the main objective of CYPHER. We will study the connection between microcystin synthesis and the cellular redox changes generated by variations in photosynthesis and in photoprotective mechanisms in MCs producer Planktothrix presenting different phenotypes and living in different ecosystems. We will also study the possible role of toxins on photosynthesis and photoprotective mechanisms under stress conditions. The originality of our proposition is to put together experts in: ecology, toxin synthesis, toxin modification and transcriptomics (partner 1), comparative genomics and secondary metabolites (partners 2) and cellular photosynthesis and photoprotective mechanisms (partner 3) to do a study that should help further predict the concentration of cyanotoxin producing strains in blooms generated by different environmental conditions, especially associated with a changing climate and a more polluted world. Thus, the expected fundamental knowledge will help ecological scientists or end-users in the large application on quality and surveys of water plans.

Infections caused by multi-drug resistant (MDR) bacteria are on the rise worldwide. Enterococcus faecalis, E. faecium and Staphylococcus aureus are major Gram-positive pathogens causing life-threatening nosocomial infections. These bacteria have developed resistance to the last resort antibiotic vancomycin and their infections are today difficult to treat. Next generation antibiotics are therefore urgently needed, especially for vancomycin-resistant E. faecium and S. aureus, which were recently ranked as high priority for the development of new antibiotics by the World Health Organization. Interesting molecules would be drugs targeting important virulence factors or resistance mechanisms without demonstrating toxicity to higher organisms. Recently we evidenced that two bacterial cyclic peptides belonging to the family of lasso peptides are promising candidates to fight MDR Gram-positive pathogens. These molecules, named sviceucin and siamycin, are able to completely abolish enterococcal virulence and vancomycin resistance, as well as S. aureus vancomycin resistance, without demonstrating toxic effects to higher animals. To our knowledge, this is the first example of molecules able to suppress virulence and antibiotic resistance, arguing that they are highly interesting molecules to study. Therefore understanding their mode of action, likely different from those of conventional antibiotics, is not only essential for their potential development as new drugs, but also will warrant the discovery of new pathways that can be targeted for antibacterial therapy. Thereby, our project aims at deciphering the underlying molecular mechanisms as well as the structure-activity relationship of sviceucin and siamycin. To achieve the goals, the highly interdisciplinary consortium will use numerous state-of-the art techniques. Based on literature data and on our thorough preliminary results, sviceucin and siamycin should have direct protein targets, i.e. histidine kinases of two-component systems. We suppose that interaction with some of these targets could trigger major changes in gene expression potentially at the basis of the observed decrease in virulence. Therefore, we will perform global transcriptomic analysis by RNA-sequencing in the presence and absence of the lasso peptides. Concerning the mechanism of the suppression of vancomycin resistance by sviceucin and siamycin, one of the strategies will be to isolate spontaneous mutants resistant to this effect of the peptides which will be analyzed by genome sequencing. This approach is a standard strategy for the identification of targets of antibacterial molecules. In addition, we will purify proteins suspected to be directly targeted by sviceucin and siamycin and analyze the interaction of these proteins with the peptide. This direct target identification will be completed by pull-down/immuno-precipitation approaches which are also complementary to the global approaches mentioned above to decipher the anti-virulence and anti-resistance mechanisms of sviceucin and siamycin. The potential targets identified will be verified using various in vivo and in vitro approaches. Based on the information obtained we will rationally design new peptides with modulated properties and examine their effects on the three pathogenic bacteria used in this study. The discovery of the potential of sviceucin or siamycin-derived peptides to fight MDR infections and their optimization in a context where the discovery of novel antibiotics is extremely rare will provide an alternative to the existing treatments against MDR Gram-positive pathogens.