Computational design and laboratory evolution of de novo oxidoreductase enzymes

在新兴的合成生物学领域的核心是一个基本目标，即构建新的功能和生物兼容的部件和设备，以并入明确的生物有机体或系统。为此，我们之前已经展示了如何在体内通过自然生化途径处理人造蛋白质，从而产生完全功能的人造c型细胞色素，而不需要进一步的体外组装。这些进化上很简单的蛋白质，Maquetes，已经被证明能够将许多天然氧化还原酶蛋白质共同的工程元素(例如可逆的O2结合，电子传递)整合到一个强大而简单的蛋白质支架中，而不是自然进化蛋白质的复杂性。C型细胞色素样块(CTM)不仅能与氧结合，还能组装新生的电子转移链、光捕获二聚体和与天然细胞色素进行蛋白质间电子转移。这个原型的进一步工程已经导致了一种CTM酶的组装，该酶能够催化天然含血红素的过氧化物酶常见的化学作用，其催化效率等于或超过当前最先进的从头酶和某些自然进化的酶。通过这项提议，我们的目标是利用我们最近在CTM酶底盘方面的成功，并使用计算(即使用MD&QM/MM方法和Rosetta设计包)和实验方法的强大组合来进化和提高这些多功能原酶的催化活性。在安德森实验室的第一次轮换中，学生将使用现有的CTM底盘创建不同的CTM文库，这些文库在坚持4-螺旋束组装和共价血红素掺入的基本原则的同时，将促进高通量筛选和定向进化策略，以整合和优化催化活性。我们将广泛使用BrisSynBio机器人套件来生成CTM文库，并在96孔板上高通量表达、纯化和筛选变异体。随后在Mulholland实验室的轮换将专注于对CTM催化途径的计算分析，确定创建底物结合位点的热点，并指示潜在地增强或改变反应活性的氨基酸取代。在这两个轮换之后，学生将返回安德森实验室，继续功能CTM酶的定向进化，同时也继续计算工作，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一个特殊机会，将对项目的进展和成功至关重要。

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.