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

BBSRC战略主题：变革性技术指导的蛋白质进化代表了当前可供选择的方法，用于增强选定的蛋白质属性或赋予其新功能，利用受自然启发的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突变来实现的-探测哪些残基在蛋白质中的不同位置是适应的。置换代表了自然进化中最常见的进化事件，可以被概念化为适应环境中微调蛋白质功能的小步骤。然而，进一步的机制在自然蛋白质进化中起着至关重要的作用，如小规模或大规模的插入/缺失(INDel)、基因复制或重组，这里称为外来进化事件(EEES)。目前，这些在定向进化活动中没有得到充分的探索，与替换相比，它们对蛋白质进化性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环境的崎岖和某些突变的非加性的阻碍，将蛋白质困在低适应度山谷中，需要反复几轮突变和筛选。我的项目旨在探索EEES是否可能帮助逃离这些适应度谷，从而由于蛋白质骨架的大幅跳跃和蛋白质骨架的自由基修饰而增强蛋白质的进化性。第二，替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，同样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indels更经常对蛋白质适合性有害，但它们更有可能产生一个改进的变体，而不仅仅是替代文库(Emond等人，2020年)。在此基础上，我的目标是确定EEE对他们快速进化模型蛋白质系统以适应新的底物特异性和催化反应的能力的影响。第三，该小组之前的工作证明了大规模蛋白质截断如何有助于早期的酶进化，在小的非活性蛋白质和功能性从头催化剂之间架起一座桥梁(Schnettler等人，2023)。更进一步说，EEES可能在从小的混杂生物催化剂到今天的大的精细调整的蛋白质的转变中发挥了更大的作用，因为由此导致的质量和序列复杂性的增加，从而增强了专门研究它们功能的能力。在这里，我的目的是了解EEES在从小的多肽生物催化剂到微调的大蛋白的早期转变中的作用。直到最近，由于这些EEES对蛋白质适合性的总体有害影响，探测这些EEES的影响是极其困难的，需要超高通量的筛选技术来捕捉罕见的改进变体。为了克服这一障碍，我的目标是使用现有的超高通量微流控筛选技术(Colin，Zinchenko和Hollfield，2015)，以及实验室开发的方法，以生成高质量的氨基酸indels蛋白库(Emond等人，2020年)。作为我的模型蛋白质系统，我将使用从头进化的极简主义阵营磷酸二酯酶(Schnettler等人，2023)，使我能够探索我项目的所有三个方面。总体而言，这个项目旨在提高我们对酶进化的各种机制特征以及EEES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.