Abstract
New strategies in organic synthesis are needed to achieve sustainability. Within the framework of green chemistry, biocatalysis is the most promising approach, but synthetic catalysis offers a wider range of reactions. Combining these two fields, as illustrated by the design of artificial enzymes, makes it possible to perform biocatalysis on non-natural products. Several approaches exist based on purified proteins or on so-called whole-cell biocatalysis in synthetic biology, but few involve the use of artificial enzymes with an artificial active site on the whole cell. The objective of the thesis written and conducted by Dr. Ismail Benhamed is the development of artificial enzymes for the oxidative cleavage of alkenes within E. coli bacteria, enabling the formation of aldehydes. These aldehydes are essential products for the perfume industry, healthcare, and as building blocks for synthetic chemistry. To achieve greater sustainability, the aldehyde precursors were chosen from biomass, particularly lignin, to enable the synthesis of vanillin, a product with high commercial value. To achieve these objectives, the artificial enzyme was synthesized by expressing or overexpressing the NikA protein, a Ni transporter in E. coli, in the different compartments of the bacterium, and by introducing an inorganic iron complex, FeL2, which has the property of moderately catalyzing the oxidative cleavage of alkenes in vitro. After a literature review outlining the state of the art in the field of artificial enzymes and synthetic biology, Chapter II describes the syntheses and characterizations of the complexes of interest and the molecules necessary for detecting vanillin and the complex within the bacterium, as well as all molecules required for analyzing the reaction. Chapter III presents the methodology for overexpressing the NikA protein in the three compartments of the E. coli bacterium. Chapter IV deals with the development of the oxidative cleavage catalysis reaction of vanillin precursor alkenes. To develop a future screening approach, a method for detecting simple vanillin formation, based on UV-visible spectroscopy, was developed. Using this method, it was possible to track aldehyde formation under various conditions (variations in the following parameters: concentrations of different reducing partners, bacteria, complexes, and enzyme localization within the bacteria), partially validating the proposed concept of (bio)catalysis. To achieve this result, several product detection methods were evaluated. The concept still needs to be extended to other alkene transformations, and especially the formation of artificial enzymes needs to be characterized in vivo using various approaches such as hyperpolarized NMR (DNP). This exploratory work demonstrates that this approach to hybrid enzymes in vivo is a promising avenue to consider in synthetic biology for the synthesis of non-natural molecules.
Aldehydes are essential building blocks found in high-value commercial products, particularly in the fragrance, food, and pharmaceutical industries. Current aldehyde production methods have limited sustainability. Therefore, within the framework of greener chemistry, more sustainable synthesis strategies for these compounds are a crucial challenge. The biocatalysis of alkene oxidative cleavage using artificial metalloenzymes is a promising and more sustainable approach for aldehyde synthesis. Within this approach, the use of artificial metalloenzymes involving an artificial active site on the whole cell remains largely unexplored. This doctoral project aims to develop an in vivo catalysis system based on artificial metalloenzymes for aldehyde synthesis from alkene oxidative cleavage within this context. To achieve this, E. coli bacteria will be transformed using recombinant plasmids that allow the overexpression of the NikA protein in various bacterial compartments. Next, an iron-based inorganic complex, FeL2, capable of moderately catalyzing the oxidative cleavage of alkenes in vitro, will be introduced to form the artificial metalloenzyme in vivo. This newly formed in vivo system will then be used to catalyze the oxidative cleavage of alkenes to produce aldehydes. To best align with green chemistry principles, precursors derived from lignin degradation will be used to valorize biomass. The in vivo catalysis of these precursors will result in the production of vanillin, an aldehyde with high economic value found in the fragrance, flavor, and pharmaceutical industries.