Elucidating and exploiting the biosynthetic pathways of natural products to produce novel pharmacologically relevant molecules
Antimicrobial resistance (AMR) poses a significant global public health threat, necessitating the discovery of new antimicrobials. Natural products (NPs) are important reservoirs for such molecules. Among them, 2,5-diketopiperazines (DKPs) stand out due to their remarkable biological activities. DKP biosynthesis typically involves a core enzyme known as cyclodipeptide synthase (CDPS), which forms a cyclodipeptide scaffold, followed by one or more tailoring enzymes that introduce chemical modifications, leading to more complex DKPs. While the diversity of DKPs obtained is substantial, it remains limited since the initial cyclodipeptide scaffolds are predominantly composed of aromatic and hydrophobic amino acids.
Recently, novel core enzymes termed RCDPSs have been identified, showing no sequence homology to CDPSs. Notably, these RCDPSs utilize aminoacyl-tRNAs as substrates to synthesize cyclodipeptide scaffolds containing arginine.
This project proposes to investigate these RCDPSs, aiming to enable the biosynthesis of diverse DKPs containing arginine and other charged amino acids. The objectives are to establish the natural repertoire of cyclodipeptide scaffolds produced by these enzymes, understand the molecular basis of their substrate specificity, and ultimately perform enzymatic and metabolic engineering to generate a broader diversity of non-natural DKPs with charged amino acids. The project will be carried out using a range of biological (molecular biology, biochemistry, biophysics) and analytical chemistry (LC-MS) methods, with collaborations involving experts in structural biology and synthetic chemistry. If the project's progress allows, a collaboration will be established with an already identified platform to test the biological activity of the generated compounds.
Trans-splicing gene therapy for Stargardt disease: construction of molecular and cellular tools to target ABCA4 gene mutations
This project aims to develop an innovative therapeutic approach for Stargardt disease, a macular degeneration caused by mutations in the ABCA4 gene. The strategy is based on SMaRT (Spliceosome-Mediated RNA Trans-splicing) technology, which enables mutation correction at the transcriptome level by replacing mutated exons of endogenous mRNA by trans-splicing with an exogenous RNA (PTM). Since the PTM contains only a part of the mRNA to be corrected, this approach can overcome the obstacle of the large size of the ABCA4 cDNA, which exceeds the carrying capacity of AAV vectors. The project will consist of several phases using molecular and cell biology techniques: construction of viral vectors for the expression of PTMs, production of cell lines to test the efficacy of binding domains (BD) to induce trans-splicing, and screening of BDs to optimize PTMs. Selected PTMs will then be tested in retinal organoids and animal models to demonstrate their therapeutic potential for the treatment of this genetic disease. As AAV is currently the most effective vector for retinal transduction, this project could open new therapeutic perspectives for Stargardt disease.
Characterization of the molecular mechanism involved in the detection of rare earth elements in Pseudomonas putida and associated biosensors development.
Rare earths (REE) are widely used in high technology, and demand for REE is set to double over the next 30 years. The selective extraction and recycling of REE has a triple challenge: economic, technological and ecological. Currently, less than 1% of REEs are recycled. What's more, extraction methods are tedious and polluting. They require several stages with acids or solvents. The discovery in 2011 of enzymes that naturally use light REE has opened up new prospects. The development of biosourced methods could be a key element in unlocking current selectivity and extraction barriers. This thesis is part of the biotechnologies of tomorrow theme. The aim of this thesis is to acquire fundamental data on the molecular mechanism of a biological system for the selective perception of REE through a robust screening, in order to take advantage of it for the development of biosensors responding to certain specific REE. Cell biology, biochemistry and in silico analysis techniques based on artificial intelligence will be used to accomplish this project. The results obtained will enable us to identify: 1) the molecular mechanism of REE detection and the factors influencing its selectivity, 2) the binding sites of the regulator and the genes involved in this response, and 3) the development from 1) and 2) of robust and selective biosensors.
New rapid diagnostic tool for sepsis: microfluidic biochip for multi-target detection by isothermal amplification
Sepsis is among the main cause of death across the world, and is caused by severe bacterial infection but can also originate from viruses, fungi or even parasites. In order to drastically increase survival rates, a rapid diagnostic and appropriate treatment is of paramount importance. The commercially available tools for nucleic acid detection by qPCR are able to sense multiple targets. However, these multiplexed analyses arise from the accumulation of analysis channels or reaction chambers where only one target can be detected. The original sample has to be divided, resulting in a loss of sensibility since a smaller amount of targets is available in channels or chambers.
In order to tackle the question of “How to detect multiple targets without a loss in sensibility?”, the PhD candidate will have to develop a multiplexed detection in a single reaction chamber by localized immobilization of LAMP primers (Loop-mediated isothermal amplification) on a solid substrate like COC or glass.
The expected outcome is a biochip allowing for real-time and fast (minutes) detection of several molecular DNA targets including: primers design and selection, primers immobilization on surface, integration of the biochip into a microfluidic cartridge and data collection and management for fluorescence detection of targets.
This innovative work will provide the PhD candidate with strong skills in diverse scientific domains such as molecular biology, surface functionalization, modelling and simulation, in a very multidisciplinary working environment.
Biogas upgrading with an advanced Biorefinery for CO2 conversion
The use of renewable energy sources is a major challenge for the coming decades. One way of meeting the growing demand for energy is to valorize waste. Among the various strategies currently developed, the recovery of biogas from anaerobic digestion plants appears to be a promising approach. Biogas is composed mainly of methane, but also of unused CO2 (around 40%). The project proposed here is to reform biogas using a renewable biohydrogen source to convert the remaining CO2 into pure CH4. We propose to set up a stand-alone advanced biorefinery that will combine photoproduction of hydrogen from waste (e.g.: lactoserum) by the bacterium Rhodobacter capsulatus combined with the CO2 present in the biogas in a biomethanation unit containing a culture of Methanococcus maripaludis, a methanogenic archaea able to produce CH4 from CO2 and H2 only (according to the Sabatier reaction). The aim is to produce CH4 in an energy-efficient and environmentally-friendly way.