Rotation 1: Mapping the evolutionary trajectories of newly evolved minimal proteins

定向蛋白质进化代表了当前增强蛋白质特性或赋予其新功能的选择方法，利用自然灵感从大量不同序列中选择改进的变体。传统上，这种多样性是通过几轮替代诱变来实现的——探测哪些残基在蛋白质的不同位置是自适应的。替代代表了自然进化中最常见的进化事件，可以被概念化为在适应度景观中微调蛋白质功能的小步骤。然而，进一步的机制在天然蛋白质进化中起着至关重要的作用，如小或大规模的插入/缺失（InDels），基因复制或重组，这里被称为外来进化事件（EEEs）。这些目前在定向进化运动中尚未得到充分的探索，与替代相比，它们对蛋白质进化能力的影响在很大程度上是未知的。首先，进化活动经常受到适应性环境的粗劣和某些突变的不可加性的阻碍，将蛋白质困在低适应性谷中，需要反复的诱变和筛选。我的项目旨在探索eee是否有助于摆脱这些适应度谷，从而提高蛋白质的可进化性，这是由于蛋白质骨干结构的巨大飞跃和根本修饰。其次，取代文库可能会阻碍生物催化剂向新底物或新反应的快速进化，这也是由于它们序列的非自由基变化。该小组之前的工作表明，虽然小规模的indel通常对蛋白质适应度有害，但它们更有可能产生改进的变体，而不仅仅是替代库（Emond等人，2020）。在此基础上，我的目标是确定eee对其快速进化模型蛋白质系统的能力的影响，以适应新的底物特异性和催化反应。第三，该小组之前的工作证明了大规模的蛋白质截断如何有利于早期的酶进化，弥合了小的无活性蛋白质和功能性从头催化剂之间的屏障（Schnettler等人，2023）。更进一步说，eee可能在从小型混杂的生物催化剂到今天的大型精细调节蛋白质的转变中发挥了更大的作用，这是由于其质量和序列复杂性的增加，从而增加了专门发挥其功能的能力。在这里，我的目标是了解eee在从小多肽生物催化剂到微调大蛋白质的早期转变中的作用。直到最近，由于这些eee对蛋白质适应度的总体不利影响，探测这些eee的影响非常困难，需要超高通量筛选技术来捕获罕见的改进变体。为了克服这一障碍，我的目标是使用现有的超高通量微流体筛选技术（Colin， Zinchenko和Hollfelder， 2015），以及实验室开发的方法来生成高质量的氨基酸InDels蛋白质文库（Emond et al., 2020）。作为我的模型蛋白质系统，我将使用de novo进化的极简cAMP磷酸二酯酶（Schnettler et al., 2023），使我能够探索我的项目的所有三个方面。总的来说，本项目旨在提高我们对酶进化的各种机制特征和eee在其中所起作用的理解。

BBSRC strategic theme: Transformative technologiesDirected protein evolution represents the current method-of-choice for enhancing select protein properties or imbuing it with novel functions, leveraging nature-inspired selection of improved variants from a large pool of diverse sequences. Traditionally, this diversity is achieved by rounds of substitution mutagenesis - probing which residues are adaptive in different positions within the protein. Substitutions represent the most common evolutionary events in natural evolution and can be conceptualised as small steps across the fitness landscape to fine-tune protein function. However, further mechanisms play a crucial role in natural protein evolution, such as small or large-scale insertions/deletions (InDels), gene duplications or recombinations, here termed exotic evolutionary events (EEEs). These are currently under-explored in directed evolution campaigns and their effect on protein evolvability compared to substitutions is largely unknown.Firstly, evolution campaigns are often hindered by the ruggedness of the fitness landscape and the non-additivity of certain mutations, trapping proteins in low-fitness valleys and requiring iterative rounds of mutagenesis and screening. My project aims to explore whether EEEs may help in escaping these fitness valleys and thus enhance protein evolvability due to the large leaps through the landscape and the radical modification of the protein backbone.Secondly, substitution libraries may hinder the rapid evolution of biocatalysts towards novel substrates or novel reactions, again due to the non-radical changes to their sequence. Previous work in the group suggested that while small-scale InDels are more often detrimental to protein fitness, they are more likely to yield an improved variant than substitution libraries only (Emond et al., 2020). Expanding on this, I aim to determine the effect of the EEEs on their ability to rapidly evolve a model protein system towards new substrate specificities and catalysed reactions.Thirdly, previous work in the group demonstrated how large-scale protein truncations may benefit early enzyme evolution, bridging the barrier between small inactive proteins and functional de novo catalysts (Schnettler et al., 2023). Going further, EEEs may have played an even bigger role in the transition from small promiscuous biocatalysts to the large finely-tuned proteins of today, due to the resulting increase in mass and sequence complexity and therefore an increased ability to specialise in their function. Here, I aim to understand the role of EEEs in the early transition from small polypeptide biocatalysts to fine-tuned large proteins.Until recently, probing the effect of these EEEs was exceedingly difficult due to their overall detrimental effect on protein fitness, requiring ultra-high-throughput screening technology to capture the rare improving variants. To overcome this barrier, I aim to use the existing ultra-high-throughput microfluidic screening technology (Colin, Zinchenko and Hollfelder, 2015), together with the methodology developed in the lab to generate high-quality protein libraries of amino acid InDels (Emond et al., 2020). As my model protein system, I will use the de novo evolved minimalist cAMP phosphodiesterase (Schnettler et al., 2023), allowing me to explore all three aspects of my project. Overall, this project aims to improve our understanding of various mechanistic features of enzyme evolution and the role the EEEs play in it.