Molecular evolution of elongation factor P in Escherichia coli

本研究旨在更好地了解延伸因子P（EF-P）的自然变异的分子事件，并探索进化的EF-P变体在大肠杆菌中异源表达含聚脯氨酸的蛋白。当需要翻译两个或更多个连续的脯氨酸时，核糖体停滞，并且EF-P被招募以减轻这种停滞。通常，EF-P仅在翻译后修饰后才有活性。例如，EF-P在生物技术应用中广泛使用的底物E中被β-赖氨酸化。大肠杆菌，其需要两种酶活性，L-赖氨酸2，3-氨基变位酶EpmB和（R）-β-赖氨酸连接酶EpmA。而E.大肠杆菌本身只有中等数量的聚脯氨酸基序，这些停滞基序的数量超过了放线菌中编码蛋白质的总数。具体而言，这些基序在次级代谢途径中高度富集，例如链霉菌属物种中的聚酮化合物脱氢酶。我们最近发现，链霉素和其他放线菌使用EF-P，它是功能性的，没有任何翻译后修饰，以补偿这种翻译负担。这种类型的EF-P的主要特征是一个刚性环（Pro-Gly-Lys-Gly-Pro），它围绕着另外修饰的氨基酸，这给了这个EF-P亚家族（PGKGP亚家族）的名字。合成生物学家能够使用即插即用的方法组装新的聚酮化合物，然而，在E.大肠杆菌低。我们将通过进化一种在大肠杆菌中没有翻译后修饰的完全功能的EF-P来解决这个问题。杆菌重要的是要注意，将刚性环引入E. coli EF-P不足以产生功能性未修饰的变体。因此，我们将使用定向进化的迭代循环，从寻找合适的“亲本”PGKGP-EF-P开始，并在efp的定点和随机突变后继续筛选越来越多的活性EF-P变体，以实现这一目标。应选择体内外活性最高的EF-P。E.表达该变体的大肠杆菌菌株应进行全面表征，并测试其在异源表达酶途径的聚酮化合物生产中的适用性。伴随着这个项目，我们将首次分析PGKGP中EF P的翻译后修饰--拟杆菌和α-变形菌的代表。这一提议不仅将扩展合成菌株工程的工具箱，还将为翻译的基本过程提供新的功能和机制见解。

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.