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

第 1 轮：绘制新进化的最小蛋白质的进化轨迹

• 批准号: 2643473
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2643473
• 关键词: Rotation; Mapping; evolutionary; trajectories; newly

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.

BBSRC战略主题：定向蛋白质进化代表了当前增强选择蛋白质特性或赋予其新功能的首选方法，利用自然启发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取代诱变来实现的-探测哪些残基在蛋白质内的不同位置是适应性的。取代代表了自然进化中最常见的进化事件，并且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，例如小规模或大规模插入/缺失（InDel），基因复制或重组，这里称为外来进化事件（EEE）。这些是目前在定向进化运动和其对蛋白质的evolvability相比，取代的效果是在很大程度上unknowed. First，进化运动往往是由健身景观和某些突变的非加和性的崎岖阻碍，捕获蛋白质在低健身谷和需要迭代轮的诱变和筛选。我的项目旨在探索EEE是否可以帮助逃离这些适应度谷，从而提高蛋白质的进化能力，因为通过景观和蛋白质骨架的激进修改的大飞跃。其次，替代库可能会阻碍生物催化剂向新底物或新反应的快速进化，同样是由于它们的序列的非激进变化。该小组先前的工作表明，虽然小规模InDel更经常对蛋白质适应性有害，但它们比仅取代文库更可能产生改进的变体（Emond et al.，2020年）。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朝向新底物特异性和催化反应的能力的影响。第三，该小组先前的工作证明了大规模蛋白质截短如何有利于早期酶进化，弥合小的无活性蛋白质和功能性从头催化剂之间的障碍（Schnettler et al.，2023年）。更进一步，EEE可能在从小型混杂生物催化剂到今天大型精细调节蛋白质的转变中发挥了更大的作用，这是由于质量和序列复杂性的增加，从而提高了专门化其功能的能力。在这里，我的目标是了解EEE的作用，在早期过渡的小多肽生物催化剂微调大protein.Until最近，探测这些EEE的影响是非常困难的，由于其整体上对蛋白质的健身不利的影响，需要超高通量筛选技术，以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体筛选技术（Colin，Zinchenko和Hollfelder，2015），以及在实验室中开发的方法来产生氨基酸InDel的高质量蛋白质文库（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最小cAMP磷酸二酯酶（Schnettler等人，#20320;，让我对这三个方面进行了探索。总的来说，这个项目旨在提高我们对酶进化的各种机制特征以及EEE在其中发挥的作用的理解。