Innovation et Biovalorisation : comment exploiter durablement les richesses de la nature
Jour 2 - 18 Avril 2024
Alexandre DELAROUZEE
Ingénierie métabolique de Clostridium acetobutylicum pour l’utilisation de sucres issus de biomasse lignocellulosique.
Les bactéries solvantogènes du genre Clostridium ont la capacité de fermenter les différents sucres extraits de la biomasse lignocellulosique afin de produire des molécules chimiques d’intérêt tel que l’acétone, le n-butanol et l’isopropanol. Elles représentent donc une alternative prometteuse dans le but de remplacer la pétrochimie par une approche biosourcée.
Cependant, la possibilité d’utiliser de telles souches au sein de procédés industriels basés sur l’utilisation de biomasse lignocellulosique est encore limitée par plusieurs aspects. L’utilisation de différents sucres par l’organisme modèle Clostridium acetobutylicum est soumise à un phénomène de répression catabolique. Le glucose étant utilisé préférentiellement par rapport aux pentoses, les rendements fermentaires obtenus lors de fermentations continues de différentes sources carbonées sont limités. De plus, le prétraitement de la biomasse lignocellulosique libère des composés toxiques pour la cellule qui vont entraver sa croissance.
Ces différentes problématiques ont été abordées au cours de cette thèse. Premièrement, des approches de mutagénèse dirigée ont permis de mieux comprendre et de moduler les mécanismes moléculaires impliqués dans le phénomène de répression catabolique. En parallèle, une approche d’évolution adaptative de C. acetobutylicum a permis d’obtenir des mutants possédant une meilleure tolérance aux composés toxiques présents dans les jus liquides de lignocellulose. Enfin, une étude de l’essentialité des gènes de C. acetobutylicum réalisée par Séquençage d’Insertion de Transposon, a donné accès à de précieuses informations sur l’organisation génétique de cet organisme modèle des Clostridium solvantogènes.
Ces résultats combinés permettront à terme de concevoir des micro-organismes possédant de meilleures capacités de fermentation de substrats issus de biomasse lignocellulosique.
Horiya Nassiba HAM
Development of an enzymatic process for alkyl hydroxycinnamatesthe synthesis
Alkyl hydroxycinnamates (AHCs) are hydroxycinnamic acid (HCA) esters aliphatic derivatives found in plants associated with suberin and cutin. The presence of the aliphatic moiety on AHCs modulates their hydrophilic/lipophilic balance and improves their integration in oil-based formulations and their biological activities compared to their phenolic acid parents. These features open a broad panel of applications for these molecules as antioxidants, antimicrobials, and UV protectants among others. Lipases are usually used to graft aliphatic moieties on phenolic acids. However, it has been reported that lipase’s activity is inhibited in the case of p-hydroxycinnamic acids due to the simultaneous presence of the p-hydroxyl and the double bound near the carboxylic function conjugated to the aromatic cycle on the phenolic acid. Our work seeks to develop an enzymatic process for the synthesis of AHC by miming their biosynthetic pathway in plants.
We expressed two recombinant enzymes in a 5L fermenter using transformed E. coli competent cells. The first one is a CoA-ligase (4CL) implicated in the activation of p-HCAs, and the second one is an acyltransferase (ACT) responsible for the transfer of the acyl moiety of the activated phenolic acid on an aliphatic moiety (acyl acceptor) for AHCs synthesis. We tested the enzymatic activity of the purified 4CL and ACT on different HCAs and acyl acceptors, respectively. HPLC and LC-MS results showed the successful activation of HCAs and AHCs synthesis. We also optimized the ATP concentration and the reaction time for HCAs activation using design of experiment. We are currently working on the investigation of ACT permissiveness using molecular docking and we aim to implement a one pot reaction and work on cofactors regeneration to minimize them as much as possible to make the process economically viable.
Clément NEVE
How to make "extreme" CPC greener: how to substitute alkanes in biphasic solvent systems using COSMO-RS Predicting Tools
Centrifugal Partition Chromatography (CPC) is a support-free chromatographic technique involving the distribution and transfer of solutes between two immiscible liquid phases according to their partition coefficient, that avoids irreversible adsorption of the analytes, allows a total recovery of injected samples and enables separations of compounds within a large polarity range. To date, there are few alternatives to alkanes when it comes to purifying highly lipophilic compounds.
However, most alkanes are classified as CMR or ecotoxic compounds. Replacing them with « greener » alternatives is an essential objective for the CPC development as an industrial-scale purification technique.
To replace alkanes, a lot of alternatives are currently being studied (oils, ethers, esters, NaDES, ionic liquids, etc..), but testing all of them in different ternary or quaternary systems, at different compositions and on various extracts is an extremely time and money-consuming approach.
COSMO-RS software is a based equilibrium thermodynamics method, placing compounds in a conductor-like environment and calculating a so-called « σ-profile », allowing to compare the similarity between two compounds (i.e., their capacity as hydrogen donor or acceptor), and thus their miscibility. This software allowed the prediction of theoretical intermolecular properties and of ternary, quaternary and solubility diagrams of biphasic solvent systems containing "green" solvents such as CPME, MeTHF, CyreneTM, vegetable oils, etc. The predicted properties were compared with experimental data and available literature. As a proof of concept, the isolation of triterpenes from Pistacia lentiscus has been achieved without using petroleum-based solvents.
Jérémy PERRET
Identifying antioxidant compounds within a mixture through dereplication and online DPPH strategies: A study on the CO2 byproduct of the Makwaen Pepper
Natural antioxidants are widely used in cosmetics, nutraceuticals, and food supplements as preservatives or to protect against reactive oxygen species. Exploring byproducts from various industries - often considered waste - presents a substantial and promising approach for developing natural and sustainable antioxidant ingredients in a circular economy perspective.
This study aimed to develop a robust and sensitive online screening workflow to identify compounds with anti-free radical activity in chemically complex natural extracts. Compared to previously reported results, the response's sensitivity and resolution were significantly improved by introducing centrifugal partition chromatography as a first separation dimension before the online workflow HPLC-DPPH-UV-HRMS2. The dereplication step was then performed by combining NMR and MS data to improve the annotation confidence level and finally merged with the free-radical activity information.
As a proof of concept, the developed original and efficient workflow was performed on the CO2 byproducts of the Makwaen pepper (Zanthoxylum myriacanthum), known for its medicinal properties and culinary seasoning. While CO2 extract of pepper fruits has gained commercial attention, particularly in the fragrance industry for their sulphury fruity olfactive profile, non-volatile compounds remain insufficiently explored.
The results were visualized as a multi-informative molecular network by matching annotated compounds with a reliability level score and anti-free radical activity activities. For instance, hydroquinone, isoquercetin, hyperoside, dicaffeoylquinic acids, chlorogenic acids and lignan glucosides were annotated and identified to be responsible for the activity of CPC fractions