Molecular evolution of elongation factor P in Escherichia coli

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This research project aims to better understand the molecular events of the natural variation of elongation factor P (EF-P) and to explore an evolved EF-P variant for the heterologous expression of polyproline-containing proteins in Escherichia coli. Ribosomes stall, when two or more consecutive prolines need to be translated, and EF-P is recruited to alleviate this arrest. Usually, EF-P is only active after post-translational modification. For example, EF-P is ß-lysinylated in the widely used chassis for biotechnological applications E. coli, which requires two enzymatic activities, the L-lysine 2,3-aminomutase EpmB and the (R)-ß-lysine ligase EpmA. While E. coli itself has only a moderate number of polyproline-motifs, the number of these stalling motifs exceeds the total number of encoded proteins in Actinobacteria. Specifically, these motifs are highly enriched in secondary metabolite pathways, such as polyketide synthases in Streptomyces species. We have recently discovered that Streptomycin and other Actinobacteria use an EF-P that is functional without any post-translational modification to compensate this translational burden. The main feature of this type of EF-P is a rigid loop (Pro-Gly-Lys-Gly-Pro) that surrounds the otherwise modified amino acid, which gives the name to this EF-P subfamily (PGKGP-subfamily). Synthetic biologists are able to assemble new polyketides using plug-and-play methods, however, heterologous expression of the corresponding pathways in E. coli is low. We will address this problem by evolving an EF-P that is fully functional without post-translational modification in E. coli. It is important to note that the introduction of the rigid loop into E. coli EF-P is not sufficient to generate a functional unmodified variant. Therefore, we will use iterative cycles of directed evolution, starting with the search of an amenable "parent" PGKGP-EF-P, and continuing with screening of more and more active EF-P variants after site-directed and random mutagenesis of efp in order to achieve this goal. EF-P with the highest activity in vivo and in vitro shall be the selected. E. coli strains expressing this variant shall be comprehensively characterized and tested for suitability in polyketide production of heterologously expressed enzyme pathways. Accompanying this project, we will analyse for the first time the post-translational modification of EF P in PGKGP-representatives of the Bacteriodetes and alpha-proteobacteria. This proposal will not only extend the toolbox for synthetic strain engineering, but will also provide new functional and mechanistic insights into the fundamental process of translation.