Mutagenesis and selection of enzymatic catalysts for biotechnological applications: development of an integrated in vivo method
Due to their properties as catalysts producing highly enantio- and regioselective compounds from target substrates under mild reaction conditions, the use of enzymes in biotechnological processes is rising. However, their often insufficient activity on non-natural compounds and narrow substrate ranges still limit their use in industrial setups. To obtain enzymes with enhanced activities, methods of directed evolution are available, involving mutant gene library generation and high throughput testing of individual variants in a cellular context. Linking of the targeted enzymatic activity to cell growth by constructing strains conditionally auxotrophic for essential metabolites or for energy carriers have significantly enlarged the application range of directed evolution (Chen et al., 2022). To achieve spatial and temporal connection between mutagenesis and variant screening, in vivo mutagenesis approaches have recently been developed. Among them are inducible systems employing different deaminase base editors tethered to T7 RNA polymerase (T7 RNAP), provoking base substitutions concomitant to transcription depending on the deaminase used (Cravens et al., 2021; (https://2021.igem.org/ Team:Evry_Paris-Saclay). However, these techniques have not yet been applied for the amelioration of industrial biocatalysts.
The components of the systems, i.e. target genes, T7 RNAP-deaminase fusion proteins and regulatory modules, are plasmid borne. The PhD student will further develop this method by inserting the T7 RNAP editor and the target gene into the E. coli chromosome, thus stabilizing the system and opening the possibility of multiple rounds of mutagenesis and selection steps in GM3 automated continuous culture devices available in the laboratory. He/she will establish a mutagenesis and selection protocol, using a native gene enabling conditional metabolic selection as reporter. The validated protocol will subsequently be applied to heterologous NADPH-dependent dehydrogenases using a generic NADPH sensor selection strain constructed and used in the lab (Lindner et al., 2018). These will include the screening for alcohol and amine dehydrogenases, activities already studied by our group (Ducrot et al., 2020), to obtain variants with broadened substrate specificity. Their potential for synthetic applications will be assessed in laboratory scale, using targets chosen in collaboration with national and international partners. In vitro characterization of the enzymatic activity of enhanced variants will also be undertaken. The PhD student will benefit from multiple expertise and equipment of the UMR Génomique Métabolique, covering molecular genetics, synthetic biology, directed evolution, chemical analytics and enzymology.
Elucidation of the homarine degradation pathway in the oceans
Context:
Primary biological production in the oceans exerts significant control over atmospheric CO2. Every day, phytoplankton transform 100 million tonnes of CO2 into thousands of different organic compounds (1). Most of these molecules (as metabolites) are biologically labile and converted back into CO2 within a few hours or days. The climate-carbon feedback loops mediated by this reservoir of labile dissolved organic carbon (DOC) depend on this network of microbes and metabolites. In other words, the resilience of the ocean to global changes(such as temperature rise and acidification) will depend on how this network responds to these perturbations.
Because of its short lifespan, this pool of labile DOC is difficult to observe. Yet these microbial metabolites are the most important carbon transport pathways in the ocean and are assimilated by marine bacteria as sources of carbon and energy. Knowledge of the main metabolic pathways (from genes to metabolites) is therefore essential for modelling carbon flows in the oceans. However, the diversity of these molecules remains largely unexplored and many of them have no annotated biosynthetic and/or catabolic pathways. This is the case for homarin (N-methylpicolinate), an abundant compound in the oceans. Homarine content can reach 400 mM in the marine cyanobacterium Synechococchus (2) and this ubiquitous organism contributes between 10 and 20% of global net primary production (3).Because of its abundance, homarine is probably an important metabolite in the carbon cycle.
Project:
In this thesis project, we aim to elucidate the homarine degradation pathway in the oceans.
Ruegeria pomeroyi DSS-3 is a Gram-negative aerobic bacterium and a member of the marine Roseobacter clade. Its close relatives account for around 10-20% of the bacterial plankton in the mixed coastal and oceanic layer (4). In the laboratory, DSS-3 can use homarine as its sole carbon source but to date, there is no information on the genes and catabolites involved in this process.
Comparative analysis of RNAseq experiments conducted on DSS-3 cultures grown with homarine or glucose (control) as a carbon source will enable us to identify the candidate genes involved in the degradation pathway. This pathway will also be studied using a metabolomic approach based on liquid chromatography coupled with very high resolution mass spectrometry. The difference in profile between DSS-3 metabolomes from cells grown on glucose as a carbon source and those from cells grown on homarine will help to detect catabolites in the pathway. Finally, the candidate genes will be cloned for recombinant expression in E. coli, the corresponding proteins purified and their activity characterized in order to reconstruct the entire homarine degradation pathway in vitro.
Analysis of the expression of these genes in data from the Tara Oceans project (5) will be the first step towards a better understanding of the role of homarine in the carbon cycle.
References :
(1) doi.org/10.1038/358741a0
(2) doi.org/10.1128/mSystems.01334-20
(3) doi.org/10.1073/pnas.1307701110
(4) doi.10.1038/nature03170
(5) https://fondationtaraocean.org/expedition/tara-oceans/
Wetting dynamics at the nanoscale
Wetting dynamics describes the processes involved when a liquid spreads on a solid surface. It's an ubiquitous phenomenon in nature, for example when dew beads up on a leaf, as well as in many processes of industrial interest, from the spreading of paint on a wall to the development of high-performance coating processes in nanotechnology. Today, wetting dynamics is relatively well understood in the case of perfectly smooth, homogeneous model solid surfaces, but not in the case of real surfaces featuring roughness and/or chemical heterogeneity, for which fine modeling of the mechanisms remains a major challenge. The main goal of this thesis is to understand how nanometric roughness influences wetting dynamics.
This project is based on an interdisciplinary approach combining physics and surface chemistry. The PhD student will conduct systematic model experiments, combined with multi-scale visualization and characterization tools (optical microscopy, AFM, X-ray and neutron reflectivity, etc.).
Thanks to the complementary nature of the experimental approaches, this thesis will provide a better understanding of the fundamental mechanisms of energy dissipation at the contact line, from the nanometric to the millimetric scale.
Understanding the signals emitted by moving liquids
Elasticity is one of the oldest physical properties of condensed matter. It is expressed by a constant of proportionality G between the applied stress (s) and the deformation (?): s = G.? (Hooke's law). The absence of resistance to shear deformation (G' = 0) indicates liquid-like behavior (Maxwell model). Long considered specific to solids, shear elasticity has recently been identified in liquids at the submillimeter scale [1].
The identification of liquid shear elasticity (non-zero G') is a promise of discoveries of new solid properties. Thus, we will explore the thermal response of liquids [2,3], exploit the capacity of conversion of mechanical energy into temperature variations and develop a new generation of micro-hydrodynamic tools.
At the nanoscopic scale, we will study the influence of a solid surface in contact with the liquid. It will be a question of studying by unique methods such as Inelastic Neutron Scattering and Synchrotron radiation, the dynamics of the solid-liquid interface using Very Large Research Facilities such as the ILL or the ESRF, as well as by microscopy (AFM). Finally, we will strengthen our collaborations with theoreticians, in particular with K. Trachenko of the Queen Mary Institute "Top 10 Physics World Breakthrough" and A. Zaccone of the University of Milan.
The PhD topic is related to wetting, macroscopic thermal effects, phonon dynamics and liquid transport.
From Combustion to Astrophysics: Exascale Simulations of Fluid/Particle Flows
This thesis focuses on the development of advanced numerical methods to simulate fluid-particle interactions in complex environments. These methods, initially used in industrial applications such as combustion and multiphase flows, will be enhanced for integration into simulation codes for exascale supercomputers and adapted to meet the needs of astrophysics. The objective is to enable the study of astrophysical phenomena such as the dynamics of dust in protoplanetary disks and the structuring of dust in protostars and the interstellar medium. The expected outcomes include a better understanding of planetary formation mechanisms and disk structuring, as well as advancements in numerical methods that will benefit both industrial and astrophysical sciences.
Mesure de la réponse intra-pixel de détecteur infrarouge à base de HgCdTe avec des rayons X pour l’astrophysique
In the field of infrared astrophysics, the most commonly used photon sensors are detector arrays based on the HgCdTe absorbing material. The manufacturing of such detectors is a globally recognized expertise of CEA/Leti in Grenoble. As for the Astrophysics Department (DAp) of CEA/IRFU, it holds renowned expertise in the characterization of this type of detector. A key characteristic is the pixel spatial response (PSR), which describes the response of an individual pixel in the array to the point-like generation of carriers within the absorbing material at various locations inside the pixel. Today, this detector characteristic has become a critical parameter for instrument performance. It is particularly crucial in applications such as measuring galaxy distortion or conducting high-precision astrometry. Various methods exist to measure this quantity, including the projection of point light sources and interferometric techniques. These methods, however, are complex to implement, especially at the cryogenic operating temperatures of the detectors.
At the DAp, we propose a new method based on the use of X-ray photons to measure the PSR of infrared detectors. By interacting with the HgCdTe material, the X-ray photon generates carriers locally. These carriers then diffuse before being collected. The goal is to derive the PSR by analyzing the resulting images. We suggest a two-pronged approach that integrates both experimental methods and simulations. Data analysis methods will also be developed. Thus, the ultimate objective of this thesis is to develop a new, robust, elegant, and fast method for measuring the intra-pixel response of infrared detectors for space instrumentation. The student will be based at the DAp. This work also involves collaboration with CEA/Leti, combining the instrumental expertise of the DAp with the technological knowledge of CEA/Leti.
Development and characterization of a reliable 13.5 nm EUV OAM carrying photon beamline
The Extreme UltraViolet (EUV) photon energy range (10-100 nm) is crucial for many applications spanning from fundamental physics (attophysics, femto-magnetism) to applied domains such as lithography and nanometer scale microscopy. However, there are no natural source of light in this energy domain on Earth because photons are strongly absorbed by matter, requiring thus vacuum environment. People instead have to rely on expensive large-scale sources such as synchrotrons, free electron lasers or plasmas from large lasers. High order laser harmonic generation (HHG), discovered 30 years ago and recognized by the Nobel Prize in Physics in 2023, is a promising alternative as a laboratory scale EUV source. Based on a strongly nonlinear interaction between an ultrashort intense laser and an atomic gas, it results in the emission of EUV pulses with femto to attosecond durations, very high coherence properties and relatively large fluxes. Despite intensive research that have provided a clear understanding of the phenomenon, it has up to know been mostly limited to laboratories. Breaching the gap towards applied industry requires increasing the reliability of the beamlines, subjects to large fluctuations due to the strong nonlinearity of the mechanism, and developing tools to measure and control their properties.
CEA/LIDYL and Imagine Optic have recently joined their expertise in a join laboratory to develop a stable EUV beamline dedicated to metrology and EUV sensors. The NanoLite laboratory, hosted at CEA/LIDYL, is based on a high repetition rate compact HHG beamline providing EUV photons around 40eV. Several EUV wavefront sensors have been successfully calibrated in the past few years. However, new needs have emerged recently, resulting in the need to upgrade the beamline.
The first objective of the PhD will be to install a new HHG geometry to the beamline to enhance its overall stability and efficiency and to increase the photon energy to 92eV, a golden target for lithography. He will then implement the generation of a EUV beam carrying orbital angular momentum and will upgrade Imagine Optic’s detector to characterize its OAM content. Finally, assisted by Imagine Optic engineers, he will develop a new functionality to their wavefront sensors in order to enable large beam characterization.
Advanced characterization of ferroelectric domains in hafnia-based thin films
Les mémoires ferroélectriques à accès aléatoire (FeRAM en anglais) à base d'oxyde d’hafnium et de zirconium (HZO) sont intrinsèquement ultra-faibles en consommation grâce au mécanisme de changement de tension, au potentiel de mise à l'échelle du HZO en dessous de 10 nm et à la compatibilité CMOS complète. De plus, elles présentent une faible latence nécessaire à une grande variété d'applications de logique et de mémoire. La compréhension des mécanismes sous-jacents et de la cinétique du ‘switching’ des domaines ferroélectriques est essentielle pour une conception intelligente des FeRAMs avec des performances optimales.
Cette thèse porte sur la caractérisation complète des domaines ferroélectriques (FE) dans des films HZO ultra-minces. L'étudiant utilisera plusieurs techniques d'imagerie de surface (microscopie à force piézoélectrique, PFM, microscopie électronique à basse énergie, LEEM, et microscopie électronique à photoémission de rayons X, PEEM) combinées à des méthodes avancées de caractérisation operando (détection résolue dans le temps couplée au rayonnement synchrotron). Ce projet marquera une avancée importante dans la recherche fondamentale des mécanismes de basculement de polarisation des couches FE ultra-minces à base d'hafnium, en élucidant les effets spécifiques de l'interface électrode métallique/couche FE dans le comportement électrostatique des condensateurs étudiés. Il permettra à terme une avancée significative dans le développement industriel des mémoires émergentes ferroélectriques, essentielles pour les applications d'intelligence artificielle (IA) à grande échelle.
First observations of the TeV gamma-ray sky with the NectarCAM camera for the CTA observatory
Very high energy gamma-ray astronomy is a relatively young part of astronomy (30 years), looking at the sky above 50 GeV. After the success of the H.E.S.S. array in the 2000s, an international observatory, the Cherenkov Telescope Array (CTA), should start operating by 2026. This observatory will include a total of 50 telescopes, distributed on two sites. IRFU is involved in the construction of the NectarCAM, a camera intended to equip the "medium" telescopes (MST) of CTA. The first NectarCAM (of the nine planned) is being integrated at IRFU and will be shipped on site in 2025. Once the camera is installed, the first astronomical observations will take place, allowing to fully validate the functioning of the camera. The thesis aims at finalizing the darkroom tests at IRFU, preparing the installation and validating the operation of the camera on the CTA site with the first astronomical observations. It is also planned for the student to participate in H.E.S.S. data analysis on astroparticle topics (search for primordial black holes, constraints on Lorentz Invariance using distant AGN).
Influence of ionization density in water on fluorescent solutes. Application to the detection of alpha radiation
The location and rapid identification, at a distance, of sources of alpha and beta particle emissions on surfaces or in wet cavities or solutions, in nuclear facilities undergoing decommissioning or to be cleaned up, is a real challenge.
The aim of the proposed PhD project is to develop a concept for the remote detection of fluorescence light from water radiolysis processes on molecules or nano-agents. Temporal characterization using fluorescence lifetime measurements will enable detection to be attributed to a type of radiation, depending on its linear energy transfer (LET). In the Bragg peak of alpha radiation, where the TEL is maximal, the ionization density due to this TEL influences the fluorescence lifetime. However, dose rate effects also need to be considered.
Molecules and nanoparticles that are candidates for forming fluorescent products and are sensitive to the ionization density and radicals produced in traces at very short times will be identified by guided bibliography work, then tested and compared by measurements. Spectral measurements (absorption and fluorescence) and fluorescence lifetimes of the corresponding fluorescent species will be carried out using the multi-channel (16-channel) TCSPC (Time Corelated Single Photon Counting) method. Ion beams or alpha particles from sealed sources will be used for proof-of-concept. Ion beams or alpha particles from sealed sources will be used for proof-of-concept in the CEA clean-up/dismantling program.