Computational design and laboratory evolution of de novo oxidoreductase enzymes

从头氧化还原酶的计算设计和实验室进化

• 批准号: 1788487
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1788487
• 关键词: Computational; design; laboratory; evolution; de

At the core of the emerging field of Synthetic Biology lies a fundamental goal to construct new functional and bio-compatible parts and devices for incorporation into explicitly biological organisms or systems. To this end, we have previously demonstrated how manmade proteins can be processed through natural biochemical pathways in vivo to produce fully functional manmade c-type cytochromes without the need for further in vitro assembly. These evolutionarily naive proteins, maquettes, have proven capable of incorporating many engineering elements common to natural oxidoreductase proteins (e.g. reversible O2 binding, electron transport) within a robust but simple protein scaffold lacking the complexity of naturally evolved proteins. The prototype c-type cytochrome maquette (CTM) is capable not only of binding oxygen, but also of assembling nascent electron transfer chains, photoactive light harvesting dyads and engaging in interprotein electron transfer with natural cytochromes. Further engineering of this prototype has resulted in the assembly of a CTM enzyme capable of catalyzing chemistries common to natural heme-containing peroxidase enzymes with catalytic efficiencies equaling or surpassing those of the current state-of-the-art de novo enzymes and certain naturally evolved enzymes.With this proposal, we aim to capitalize on our recent successes with the CTM enzyme chassis and use a powerful combination of computational (i.e. using MD & QM/MM methodologies and the Rosetta design package) and experimental approaches to evolve and improve catalytic activity within these versatile protozymes. During the first rotation in the Anderson lab, the student will use the existing CTM chassis to create diverse CTM libraries that, while adhering to the basic principles of 4-helix bundle assembly and covalent heme incorporation, will facilitate high throughput screening and directed evolution strategies to incorporate and optimize catalytic activity. We will extensively use the BrisSynBio robotics suite to generate CTM libraries and for the high-throughput expression, purification and screening of variants in 96-well plates. The subsequent rotation in the Mulholland lab will focus on the computational analysis of the CTM catalytic pathway, identifying hotspots for creating substrate-binding sites, and indicating amino acid substitutions that will potentially enhance or alter reactivity. Following these two rotations, the student will return to the Anderson lab to continue the directed evolution of functional CTM enzymes while also continuing the computational work to aid redesign and analysis. This synthesis of computational and experimental techniques is an exceptional opportunity for postgraduate training and will be vital to the progression and success of the project.

合成生物学新兴领域的核心是一个基本目标，即构建新的功能和生物相容性部件和设备，以纳入明确的生物有机体或系统。为此，我们以前已经证明了如何人造蛋白质可以通过天然的生物化学途径在体内加工，以产生功能齐全的人造c型细胞色素，而不需要进一步的体外组装。这些进化上幼稚的蛋白质，maquettes，已被证明能够将许多天然氧化还原酶蛋白质常见的工程元件（例如可逆的O2结合，电子传递）整合在一个强大但简单的蛋白质支架内，缺乏天然进化蛋白质的复杂性。原型C型细胞色素模型（CTM）不仅能够结合氧，而且还能够组装新生电子传递链，光敏捕光二联体，并与天然细胞色素进行蛋白质间电子传递。该原型的进一步工程化已经导致能够催化天然含血红素过氧化物酶共有的化学的CTM酶的组装，其催化效率等于或超过当前最先进的从头酶和某些天然进化的酶的催化效率。我们的目标是利用我们最近在CTM酶底盘上的成功，并使用强大的计算组合，（即使用MD & QM/MM方法和Rosetta设计包）和实验方法来进化和改进这些多功能的原酶内的催化活性。在安德森实验室的第一轮，学生将使用现有的CTM底盘创建不同的CTM库，同时坚持4-螺旋束组装和共价血红素掺入的基本原则，将促进高通量筛选和定向进化策略，以纳入和优化催化活性。我们将广泛使用SynBio机器人套件来生成CTM文库，并在96孔板中进行高通量表达，纯化和筛选变体。穆赫兰实验室的后续轮换将专注于CTM催化途径的计算分析，确定用于创建底物结合位点的热点，并指示可能增强或改变反应性的氨基酸取代。在这两次轮换之后，学生将返回安德森实验室继续功能性CTM酶的定向进化，同时继续计算工作以帮助重新设计和分析。这种计算和实验技术的综合是研究生培训的绝佳机会，对项目的进展和成功至关重要。