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策略主题：转化技术指导的蛋白质进化代表了当前的选择方法，用于增强精选蛋白质特性或将其赋予新功能，利用自然启发的从大量不同序列中选择改进的变体。传统上，这种多样性是通过替代诱变的一轮探测来实现的 - 残基在蛋白质内的不同位置具有适应性。替代是自然进化中最常见的进化事件，可以概念化为在整个健身景观中以微调蛋白质功能的小步骤。然而，进一步的机制在自然蛋白的演化中起着至关重要的作用，例如小型或大型插入/缺失（Indels），基因重复或重组，在这里称为外来进化事件（EEES）。这些目前在有指导性的进化运动中探讨了，并且与替代相比，它们对蛋白质可变性的影响在很大程度上是未知的。高低的进化运动通常是由于健身景观的坚固性和某些突变的非添加性而阻碍了某些突变的不可吸收性，而在低位谷中捕获了蛋白质，并需要在低位级别和需要进行均匀的综合综合综合综合综合。我的项目旨在探索EEES是否可以帮助逃脱这些健身谷，从而增强蛋白质的可变性，这是由于景观的大飞跃和蛋白质骨架的根本性修改。替代图书馆可能会促进生物催化剂对新型材料反应或新颖的反应的快速演变，从而再次因其序列而变化，以使他们的序列更改为非序列。该小组的先前工作表明，虽然小规模的indels更经常不利于蛋白质适应性，但与仅替代文库相比，它们更有可能产生改善的变体（Emond等，2020）。为此，我的目的是确定EEE对它们快速发展模型蛋白质系统的能力的影响，以新的底物特异性和催化反应。三分之二的是，该小组的先前工作证明了大规模蛋白质的截断如何使早期的酶进化受益，从而使小型非活性蛋白蛋白和功能性蛋白质和功能性cataly catalys（Schnetters）（Schnetters）（Schnetters）（Schnetters）（Schnetter）（Schnetters）弥补。进一步，由于质量和序列复杂性的增加，EEE在从小型混杂生物催化剂到当今大型精细调整的蛋白质的过渡中可能发挥了更大的作用，这是由于质量和序列复杂性的提高，因此提高了专门从事其功能的能力。在这里，我的目标是了解EEE在早期从小多肽生物催化剂到微调大蛋白的早期过渡。直到最近，由于它们对蛋白质适应性的总体有害作用，需要超高的筛选技术来捕获稀有改进的变异，因此探测这些EEES的影响非常困难。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查技术（Colin，Zinchenko和Hollfelder，2015年），以及在实验室中开发的方法论，以生成氨基酸indels的高质量蛋白质库（Emond et al。，2020）。作为我的模型蛋白质系统，我将使用从头发展的简约磷酸二酯酶（Schnettler等，2023），使我能够探索项目的所有三个方面。总体而言，该项目旨在提高我们对酶进化的各种机械特征的理解以及EEES在其中所扮演的作用。