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/
Study of the links between the dysregulations of metabolism and epigenetics marks in Huntington’s disease
We want to focus on epigenetic dysregulation in Huntington’s Disease (HD), a pathogenic mechanism implicated in accelerated aging of striatal neurons. Specifically, we will investigate the interplay between altered energy metabolism and epigenetic impairment in HD striatal neurons to identify new targets/pathways for disease-modifying intervention. We aim to obtain detailed maps of histone post-translational modifications (PTMs), especially of methylations, acetylation and the recently described lactylation, which might be critical in the HD brain. Indeed, these PTMs are tightly regulated by the metabolic status of the cells. We will use proteomics which is the best suited approach to identify and quantify multiple protein PTMs. We consider working on the striatum of WT, R6/1 transgenic mice and the more progressive Q140 knock in model at various stages of disease, to assess evolution of histone PTMs and metabolism with aging. Additionally, to get a dynamic view of the links between metabolic and epigenetic imbalance in HD, we will inject intraperitoneally HD mice and controls with 13C-glucose and analyze over a time course the incorporation of 13C into histone PTMs. Finally, acetyl-CoA, the precursor for histone lysine acetylation, has been shown to be locally produced in the nucleus, by either acetyl-CoA synthetase 2 (ACSS2), ATP-citrate lyase (ACLY) or the pyruvate dehydrogenase complex. Regarding lactylation, it is currently unknown where, and by which enzymes, the pool of lactate used for modifying histone lysines by lactylation is produced. ACSS2 is a very good candidate, as it can catalyze the production of acyl-CoA molecules from the corresponding short chain fatty acids (SCFA). To address the question of the production of metabolites in the vicinity of chromatin in striatal cells, we will use epigenomics (ChIPseq or CUT&tag) to get the genomic distribution of ACSS2 and ACLY and compare it to distributions of acetyl and lactyl histone marks.
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.