Exploring linker's sequence diversity in protein fusion for the biosynthesis of zeaxanthin. | Exploration de la diversité de séquence des linkers dans les protéines de fusion pour la biosynthèse de zéaxanthine.

English. About 70% of the proteins consist of a covalent assembly of catalytic modules (multidomain proteins) and their functions depend on these associations. In addition, the concept of metabolon, introduced in the 1980s, shows the importance of spatial clustering of enzymes involved in the same biosynthetic pathway and has been applied to metabolic engineering since the early 2010s. The rationalization of the design of these enzymatic complexes is, at the present time, rather undeveloped. We have recently demonstrated that fusion proteins between 3 enzymes of the carotenoid pathway are not only able to increase the yield of lycopene or B-carotene in the yeast S. cerevisiae, but can also guide the metabolic flux to the production of minority metabolites. The Singaporean partner has recently developed an approach to engineering the carotene pathway in E. coli by subdividing the metabolic pathway into sequential modules. The transcriptional engineering of each module allows a first selection of the best assemblies, refined then by an internal engineering of each winning module. For this PhD subject, we plan to group our strengths in a study using several enzymes involved in the carotenoids pathway, with various type of assembly, and expressed in the two host microorganisms. The three main objectives are (i) to define the characteristics of the most efficient catalytic assembly, (ii) to compare catalytic architecture and metabolic flux toward particular intermediates, (iii) to increase the metabolic flux by capturing the product of the reaction. The project is original on many levels. First of all, there are few studies that compare the efficiency of catalytic domain assembly strategies with natural or fully synthetic linkers and test the genericity of these systems in a prokaryotic or eukaryotic host. Moreover, there is still no system for capturing the product (s) of this type of metabolic pathway. Finally, this research program will bring together a panel of multidisciplinary techniques (physicochemical methods, structural analysis, genetic engineering, protein engineering) to define the concepts of assembling biocatalysts.

French. La production de molécules d'intérêt par des voies biotechnologiques est une alternative écologiquement viable à la chimie. Pour cela, les récentes avancées de la biologie de synthèse fournissent des outils conceptuels et technologiques innovants, notamment dans les domaines de l'ingénierie métabolique. Cependant, une des limitations possibles de l'ingénierie métabolique est le faible rendement de production, en partie dû à une mauvaise organisation spatiale des enzymes de la voie de production dans des systèmes synthétiques. Dans d'un partenariat avec l'unité A*STAR de Singapour, nous souhaitons initier une collaboration autour de la production de caroténoïdes, molécules à fort potentiel économique en agroalimentaire et dans le domaine de la santé. Le projet vise à comprendre et maîtriser l'optimisation de la voie des caroténoïdes dans deux organismes à forts potentiels industriels : la levure Saccharomyces cerevisiae et la bactérie Escherichia coli.